doi
stringlengths 13
36
| title
stringlengths 37
233
| abstract
stringlengths 0
9.8k
| fulltext_sections
listlengths 1
127
| fulltext_additional
listlengths 0
17
|
|---|---|---|---|---|
10.3389/fbioe.2022.816362
|
Sa12b Improves Biological Activity of Human Degenerative Nucleus Pulposus Mesenchymal Stem Cells in a Severe Acid Environment by Inhibiting Acid-Sensitive Ion Channels
|
<jats:p>Sa12b is a wasp peptide that can inhibit acid-sensitive ion channels (ASICs). The biological effects of nucleus pulposus mesenchymal stem cells (NP-MSCs) have not been investigated. Therefore, this study investigated the effect of Sa12b on the biological activity of NP-MSCs through ASICs in the acidic environment of intervertebral disc degeneration (IVDD). In this study, NP-MSCs were isolated from the nucleus pulposus (NP) in patients who underwent lumbar disc herniation surgery, identified by flow cytometry and tertiary differentiation, and cultured <jats:italic>in vitro</jats:italic> in an acidic environment model of IVDD with a pH of 6.2. Proliferation, and apoptosis were observed after different Sa12b concentrations were added to P2 generation NP-MSCs. The Ca<jats:sup>2+</jats:sup> influx was detected using flow cytometry and laser confocal scanning microscopy, and qPCR was used to detect the relative expression of stem cell–associated genes (Oct4, Nanog, Jag1, and Notch1), the relative expression of extracellular matrix (ECM)–associated genes (collagen II, aggrecan, and SOX-9), and the relative expression of genes encoding ASICs (ASIC1, ASIC2, ASIC3, and ASIC4). Western blotting was used to detect the protein expression of collagen II and aggrecan in different treatment groups. Cells isolated and cultured from normal NP were spindle-shaped and adherent, and they exhibited expansion <jats:italic>in vitro</jats:italic>. Flow cytometry results showed that the cells exhibited high expression of CD73 (98.1%), CD90 (97.5%), and CD105 (98.3%) and low expression of HLA-DR (0.93%), CD34 (2.63%), and CD45 (0.33%). The cells differentiated into osteoblasts, adipocytes, and chondrocytes. According to the International Society for Cellular Therapy criteria, the isolated and cultured cells were NP-MSCs. With an increase in Sa12b concentration, the cell proliferation rate of NP-MSCs increased, and the apoptosis rate decreased significantly, reaching the optimal level when the concentration of Sa12b was 8 μg/μl. When the Sa12b concentration was 8 μg/μl and contained the ASIC non-specific inhibitor amiloride, the Ca<jats:sup>2+</jats:sup> influx was the lowest, followed by that when the Sa12b concentration was 8 μg/μl. The Ca<jats:sup>2+</jats:sup> influx was the highest in the untreated control group. qPCR results showed that as the concentration of Sa12b increased, the relative expression of Oct4, Nanog, Jag1, Notch1, collagen II, aggrecan, and SOX-9 increased, while that of ASIC1, ASIC2, ASIC3, and ASIC4 decreased. The difference was statistically significant (<jats:italic>p</jats:italic> &lt; 0.05). In conclusion, Sa12b can improve the biological activity of NP-MSCs in severely acidic environments of the intervertebral disc by reducing Ca<jats:sup>2+</jats:sup> influx <jats:italic>via</jats:italic> AISC inhibition and, probably, the Notch signaling pathway. This study provides a new approach for the biological treatment of IVDD. Inhibition of AISCs by Sa12b may delay IVDD and improve low back pain.</jats:p>
|
[
{
"section_content": "Low back pain (LBP) is one of the most common public health problems and is the leading cause of disability worldwide.It causes a severe social burden and mainly occurs among adults 25-49 years of age (Dieleman et al., 2020;O'Sullivan et al., 2020).Current treatments for LBP include mainly conservative management and surgery, which cannot cure the fundamental causes (Corp et al., 2021).According to previous reports, intervertebral disc (IVD) degeneration (IVDD) is the leading cause of LBP.In addition to standard conservative management and surgical treatment, experimental cell regeneration technology has also received extensive attention from researchers as a treatment strategy for lumbar IVDD (Kos et al., 2019). The IVD is an avascular and non-renewable tissue composed of the central nucleus pulposus (NP), peripheral fibrous annulus, and upper and lower cartilage endplates.The NP plays a central role in IVD function and consists of nucleus pulposus cells (NPCs) and extracellular matrix (ECM) components.After the degeneration of the NP, nutrient metabolism is unbalanced, and the secretion of ECM components decreases, especially that of collagen II and aggrecan.This reduces the water content in the NP, increases the osmotic pressure, decreases the pH value, induces hypoxia, and accelerates internal disc disruption (IDD).In addition, the drop in disc height eventually leads to IVDD.Therefore, regulation of NPC function and restoration of the ECM metabolic balance is necessary to delay IVDD progression (Zhang et al., 2016).Recently, several studies have shown that nucleus pulposus mesenchymal stem cells (NP-MSCs) can be regenerated and differentiated into NP-like cells in IVDD.Biological treatments, including those based on cytokines, stem cells, and other biological materials, can promote the expression of proteoglycans in the degenerated NP, increase the water content in the IVD, and thus play a role in delaying IVDD (Tao et al., 2014;Tao et al., 2015;Bowles and Setton, 2017). Relevant studies have shown that with IVDD, many changes occur in the internal environment of IDD, resulting in hypoxia, low pH, hypertonicity, and low nutrition (Colombier et al., 2014).A low pH can reduce the biological activity of NP-MSCs in IVDD.In addition, the change in pH in vivo is mainly controlled by acidsensitive ion channels (ASICs) to regulate the biological activity of cells (Vullo and Kellenberger, 2020).ASICs are gated ion channels that belong to an essential member of the epithelial sodium channel/degenerin (DEG/ENaC) family of sodiumselective channels.They are important receptors for changes in the acidic environment in vivo and are widely distributed in various body tissues.ASICs are encoded by four genes and consist of seven subunits: ASIC1a, ASIC1b, ASIClb2, ASIC2a, ASIC2b, ASIC3, and ASIC4, which are expressed in both normal and degenerative NP and are positively correlated with the degree of degeneration (Cuesta et al., 2014).In addition, studies have found that changes in the acid environment of IDD regulate the biological activity of NP-MSCs through ASICs.Human degenerative NP-MSCs express ASIC1, ASIC2, ASIC3, and ASIC4, and this expression is inversely proportional to pH (Liu et al., 2017). Carmen Hernández et al. purified and extracted the short peptide Sa12b (EDVDHVFLRF) from the venom of the solitary wasp Sphex argentatus and found that it belonged to a new type of non-specific ASIC inhibitor.Preincubation with Sa12b reversibly inhibited the amplitude of the ASIC current peak (IC50~81 nM) in rat DRG neurons in a concentration-dependent manner but had no consistent effect on the time course of desensitization or the persistent component of the current.Thus, the inhibitory effect of Sa12b (IC50 = 81 nM) on ASIC current is equivalent to that caused by peptides of plant and animal origin, such as chlorogenic acid, gastrodin, phenol, APETx2, and mambalgines (Hernández et al., 2019).In addition, related studies have shown that when the pH value of the in vivo environment changes, the opening probability, ion permeability, and ion selectivity of ASICs can be used to adjust the amount of Ca 2+ transported into cells, thereby adjusting the biological activity of cells (Zhou et al., 2016).However, it is unclear whether Sa12b mediates Ca 2+ influx through ASICs, thereby affecting the biological activity of NP-MSCs and alleviating or inhibiting IVDD.Thus, this study aimed to investigate the effect of Sa12b on the biological activity of NP-MSCs through ASICs in the acidic environment of intervertebral disc degeneration (IVDD). ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "The Sa12b mother liquor used in this study was customsynthesized at Sangon Biotech (purity >90%, Shanghai, China).The peptide sequences are presented in Table 1.This ",
"section_name": "Preparation of Sa12b Mother Liquor",
"section_num": null
},
{
"section_content": "Isolation of cells from patients with lumbar disc herniation was performed; Table 2 provides detailed information about the age, sex, and disease status of the subjects.In accordance with the Declaration of Helsinki, all procedures for the study were approved by the local ethics committees of our institution and were performed with the informed consent of the patients.After washing the sample twice with PBS (Gibco), the annulus fibrosus and cartilaginous endplate were carefully removed, and the NP tissue was cut into approximately 1 mm 3 sections and digested in 5% CO 2 at 37 °C with collagenase II solution (0.02 mg/ml).After 8 h, the cells were precipitated by centrifugation at 1,500 rpm for 5 min.The supernatant was removed, and the free cells and partially digested tissue were cultured as explants in a standard MSC expansion medium consisting of Dulbecco's modified Eagle's medium-low glucose (Hyclone, United States), 10% fetal bovine serum (FBS; Gibco, United States), and penicillin/ streptomycin (United States, Gibco) in a humidified incubator at 37 °C and 5% CO 2 .After 24 h, suspended cells and debris were removed from the medium, and the medium was completely replaced every 3-4 days to promote adherent cell growth.When the cells reached approximately 90% confluence, the culture was passaged at a ratio of 1:3.The second-generation cells were harvested using trypsin-EDTA solution (United States, Gibco) and tested. ",
"section_name": "Isolation and Culture of Cells",
"section_num": null
},
{
"section_content": "After digesting NP-MSCs with 0.25% trypsin (Biosharp, United States), the cells were washed and resuspended in 100 µl PBS (Sigma, United States) containing 1% FBS.Each tube contained 5 µl of the following antibodies, according to the recommendations of the International Society for Cell Therapy: CD34-APC, CD73-FTTC, CD45-PE, CD90-FTTC, CD105-PE, and HLA-DR-APC (eBioscience, United States).In each case, isotype control (eBioscience, United States) was used. After incubation with the antibody for 30 min at room temperature, the cells were washed with PBS, and the supernatant was discarded.The cells were resuspended in 200 ul of PBS (Sigma, United States) and analyzed using a flow cytometer (BD, United States).Immunophenotyping analysis was performed to identify the percentage of positive cells and the fluorescence intensity. ",
"section_name": "Identification of NP-MSC",
"section_num": null
},
{
"section_content": "The normal medium was replaced with an osteogenic differentiation medium (Cyagen, United States).The differentiation process lasted for 14 days, and the medium was changed every 2-3 days.After differentiation, the medium was removed, and the cells were washed with PBS and fixed with 4% paraformaldehyde solution (Sangon Biotech, China) at room temperature for 30 min.After washing twice with PBS, the cells were stained with alizarin red working solution (Cyagen, United States) for 15 min.Finally, the culture was washed three times with PBS and observed under an optical microscope. ",
"section_name": "Osteogenic Differentiation",
"section_num": null
},
{
"section_content": "NP-MSCs from the second passage were seeded in a six-well plate at a density of 5 × 104 cells/cm 2 .When the cells reached 100% confluence, the normal medium was replaced with adipogenic differentiation medium A (Cyagen, United States).After 3 days, differentiation medium A was replaced with adipogenic differentiation medium B (Cyagen, United States) for 24 h.Then, medium B was replaced with medium A again.This cycle was repeated for three to four cycles for a total of 28 days.After differentiation, the medium was removed from the wells, and the cells were washed with PBS and fixed with 4% paraformaldehyde solution for 30 min.After washing twice with PBS, the cells were stained with Oil Red O working solution (Cyagen, United States) for 30 min at room temperature.The culture was then washed three times with PBS and observed under an optical microscope. ",
"section_name": "Adipogenic Differentiation",
"section_num": null
},
{
"section_content": "NP-MSCs from the second passage were resuspended in chondrogenic differentiation basal medium (Cyagen, United States), centrifuged at 1,500 rpm for 5 min, and the supernatant was discarded.The cells were resuspended in a complete chondrogenic differentiation medium (Cyagen, United States) at a density of 5 × 105 cells/ml.Next, 500 μl of the cell suspension was placed in a 15 ml centrifuge tube containing 2.5 × 105 cells and centrifuged at 1,500 rpm for 5 min to form a pellet.The pellet was cultured at 37 °C in 5% CO 2 in a complete chondrogenic differentiation medium without interference.After 3 days, the medium was changed, and the bottom of the test tube was flicked to ensure that the cartilage ball floated freely.After that, the differentiation medium was changed every 2 days, and the differentiation process lasted for 28 days. When the diameter of the cartilage ball increased to 3 mm, tissue sections were fixed in formalin and embedded in paraffin (Sangon Biotech, China).Finally, the sections were deparaffinized and hydrated with distilled water.The sections were then stained with Alcian Blue (Cyagen, United States) for 30 min.They were washed with running tap water for 2 min and then with distilled water several times.Finally, the sections were observed under an optical microscope. ",
"section_name": "Chondrogenic Differentiation",
"section_num": null
},
{
"section_content": "Media with different pH values were prepared by adding an appropriate amount of sterilized HCl (1 mol/L) and NaOH (1 mol/L) to the culture medium and monitoring the pH values using a pH microelectrode (Ramagnetic phs-25, China).After pH values (7.4,6.2) were obtained, the medium was kept in 5% CO 2 at 37 °C for 3 days to establish a pH balance (CO 2 -dependent). ",
"section_name": "Preparation of Media With Different pH Values",
"section_num": null
},
{
"section_content": "The second-generation NP-MSCs were inoculated in 96-well plates (200 μl medium per well) at a density of 3 × 104 cells/ ml.These cells were cultured in a complete medium with a pH of 6.2 at 37 °C with 5% CO 2 .To observe the effect of Sa12b on proliferation, the experimental group also contained 20, 40, 60, and 80 µg Sa12b.Sub-samples were collected from three wells on days 1, 3, 5, 7, and 9, and 20 µl of CCK-8 (Japan, DOJINDO) was added to the cells, which were then incubated for 2 h.The isotype group did not contain any cells.The absorbance of each group was measured using a Spectrum MAX microplate reader. ",
"section_name": "Cell Proliferation Assay",
"section_num": null
},
{
"section_content": "NP-MSCs from the second passage were seeded in a 12-well plate at a density of 1 × 105 cells/ml.These cells were cultured for 2 days at different pH levels at 37 °C and 5% CO 2 .CAM and the nucleic acid dye propidium iodide (PI, Sigma, United States) were used to label live and dead NP-MSCs, respectively.Briefly, the cells were incubated with 2 μmol/L CAM and 5 μmol/L PI at room temperature for 30 min in the dark and then gently rinsed with PBS three times.A fluorescence microscope was used to collect the images. ",
"section_name": "Cell Cytotoxicity Assessment",
"section_num": null
},
{
"section_content": "NP-MSCs from the second passage were seeded in a 12-well plate at a density of 1 × 105 cells/ml.These cells were cultured for 2 days at different pH levels at 37 °C and 5% CO 2 .To observe the effect of Sa12b on cell apoptosis, the experimental group also contained Sa12b at various concentrations (2, 4, 6, and 8 μg/μl), with four replicate wells in each group.The adherent cells were collected by trypsinization without EDTA (Biosharp, United States) and washed twice with PBS.In addition, Annexin V-FITC (5 µL) and PI (KeyKey BioTECH, China) were added to each group and incubated at room temperature in the dark for 15-20 min, and flow cytometry (United States, BD) was used within 1 h to detect the percentage of apoptotic cells. ",
"section_name": "Apoptosis Measurement of NP-MSCs",
"section_num": null
},
{
"section_content": "NP-MSCs from the second passage were seeded in T-25 culture flasks at a density of 5 × 105 cells/ml and cultured in 5% CO 2 at 37 °C at different pH levels for 7, 14, and 28 days.To observe the effect of Sa12b on cell apoptosis, the experimental group also contained Sa12b at various concentrations (2, 4, 6, and 8 μg/μl).TRIzol (Ambion, United States) was used to extract total RNA according to the manufacturer's instructions, and the RNA samples were treated with DNase/RNase-free water.A Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, United States) was used to determine the quality and quantity of RNA.A reverse transcription reagent (Takara, Japan) was used to obtain cDNA from total RNA.A total of 1,000 ng of RNA was mixed with 2 µl of 5× PrimeScript RT ® MasterMix and RNase-free ddH2O (the total system volume was 10 µl).The mixed solution was first incubated at 37 °C for 15 min and then at 85 °C for 5 s, and stored at -80 °C for qPCR.All genes were analyzed by qPCR, and GAPDH was used as a control.SYBR Premix Ex Taq PCR kit (Takara, Japan) and LightCycler (Roche, Switzerland) were used for qPCR analysis.Premier software (version 5.0) was used to design the primers for all genes, as shown in Table 3. ",
"section_name": "Quantitative Real-Time PCR Analysis of Gene Expression",
"section_num": null
},
{
"section_content": "NP-MSCs from the second passage were seeded in a 6-well plate at a density of 1 × 105 cells/ml.These cells were cultured in 5% CO 2 at 37 °C at different pH levels for 14 days.To observe the effect of Sa12b on cell apoptosis, the experimental group also contained Sa12b at various concentrations (2, 4, 6, and 8 μg/μl).The cells were washed three times with ice-cold PBS, and total protein was extracted using RIPA buffer containing 1% PMSF.Protein concentration was measured using a BCA protein quantification kit (Takara, Japan).The protein was electrophoresed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Massachusetts, United States).After blocking with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 1 h, the membrane in TBST was incubated with appropriate primary antibodies: antiaggrecan antibody, anti-collagen II antibody (maintained at 4 °C overnight), and anti-GAPDH antibody (1:1,000; Santa Cruz).The membrane was then incubated with horseradish peroxidase (HRP)-labeled secondary immunoglobulin G (1: 1,000, Santa Cruz) at room temperature for 1 h.After washing the membrane three times with TBST, the immunoreactivity was detected with an enhanced chemiluminescence (ECL, Millipore) substrate, and the optical density was measured using Quantum One software (BioRad Laboratories Inc., Munich, Germany).GAPDH was used as the loading control. ",
"section_name": "Western Blot Analysis",
"section_num": null
},
{
"section_content": "NP-MSCs from the second passage were seeded at a density of 1 × 105 cells/ml in a special cell culture dish for confocal microscopy and cultured in an acidic environment (pH = 6.2) in 5% CO 2 at 37 °C.The cells were then washed three times with D-Hanks' solution and incubated with 5 μM Fluo-3-AM (Dojindo Laboratories, Japan) for 30 min at 37 °C, followed by three washes and an additional incubation in normal Hanks' solution for 15 min.Fluo-3-AM was excited at 488 nm using a laser scanning confocal microscope, and the emission was measured at 510 nm. ",
"section_name": "Calcium Imaging",
"section_num": null
},
{
"section_content": "Cell suspensions in Eppendorf tubes were washed three times with D-Hanks' solution and incubated with 5 μM Fluo-3-AM (Dojindo Laboratories, Japan) for 30 min at 37 °C.Then, the cells were washed three times for 5 min and incubated in normal Hanks' solution for 15 min.Fluo-3-AM was excited at 488 nm using a flow cytometer, and emission was measured at 510 nm. ",
"section_name": "Detection of [Ca 2+ ] i Using Flow Cytometry",
"section_num": null
},
{
"section_content": "All data were statistically analyzed using SPSS 23.0 and are presented as the mean ± standard deviation.ANOVA of the factorial design was performed to analyze the main effect and the interaction between the groups and the time periods, and oneway ANOVA was performed for multiple-group comparisons.The Student-Newman-Keuls test (homogeneity of variance) or Tamhane's test (heterogeneity of variance) was performed to compare any two groups.Statistical significance was set at p < 0.05. ",
"section_name": "Statistical Analysis",
"section_num": null
},
{
"section_content": "The Experimental Cells Were Identified as NP-MSCs Through Immunophenotyping and In Vitro Pluripotent Differentiation The cells were extracted from human IVDs, isolated, and cultured in vitro.The primary cells showed a short spindle shape after 3-5 days (Figure 1A).After approximately 2 weeks of passage, the growth rate of the primary cells was significantly faster than that of the primary cells, with spiral growth, uniform morphology, and a characteristic spindle shape (Figure 1B).Flow cytometry showed that the cultured cells had high expression of CD73, CD90, and CD105 and low expression of CD34, CD45, and HLA-DR (Figure 2A).In addition, after the cells were incubated in differentiation induction culture, strongly stained mineralized nodules indicated osteogenic differentiation, oil red O staining of intracellular lipid vacuoles indicated adipogenic differentiation, and staining with sulfated proteoglycans indicated chondrogenic differentiation (Figure 2B).In summary, the cells met the evaluation criteria of the International Society for Cellular Therapy (ISCT) for NP-MSCs. ",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "As shown in Figure 2A, compared with the normal culture environment (pH = 7.4), the proliferation of human degenerative NP-MSCs cultured in an acidic environment (pH = 6.2) was significantly inhibited (p < 0.001); however, after adding different concentrations of Sa12b (0, 2, 4, 6, 8 μg/μl) to the environment, the proliferation inhibition was significantly improved (p < 0.05), and this effect was strongest at 8 μg/μl.In addition, confocal microscopy was used to evaluate cytotoxicity in cells treated with different concentrations of Sa12b.The results indicated that Sa12b could alleviate the proliferation inhibition of NP-MSCs in a severely acidic environment without inducing cytotoxic effects (Figure 2B). ",
"section_name": "Sa12b Alleviated Proliferation Inhibition of NP-MSCs in a Severely Acidic Environment",
"section_num": null
},
{
"section_content": "Human degenerative NP-MSCs were cultured in an acidic environment (pH = 6.2) for 2 days, and different concentrations of Sa12b (0, 2, 4, 6, and 8 μg/μl) were added.Flow cytometry was used to evaluate the percentage of apoptotic NP-MSCs after treatment under the above conditions.The results showed that compared with that in the control group, the apoptosis rate of NP-MSCs gradually decreased significantly after adding Sa12b (Figures 3A,B). ",
"section_name": "Sa12b Reduced the Apoptosis Rate of NP-MSCs in a Severe Acidic Environment",
"section_num": null
},
{
"section_content": "To evaluate the effect of Sa12b on the subunits of ASICs (ASIC1, ASIC2, ASIC3, and ASIC4) and ECM-related genes (aggrecan, collagen II, and SOX-9) in NP-MSCs in an acidic environment, we used different concentrations of Sa12b (0, 2, 4, 6, and 8 μg/μl).NP-MSCs were cultured for 7, 14, and 28 days.The expression of each gene was detected by qPCR, and GAPDH expression was used as a control.The results showed that, relative to the expression levels of each gene in cells that were not treated with Sa12b, the relative gene expression of the ASIC subunits gradually decreased with the increase in Sa12b concentration (Figure 4), while that of the ECM-related genes gradually increased (p < 0.01) (Figure 5).The results also showed that these differences were maintained at least until the 28th day.In addition, western blotting results showed that the expression of ECM-related proteins (aggrecan and collagen II) in NP-MSCs also increased with an increase in Sa12b concentration, which was consistent with the qPCR results. ",
"section_name": "Sa12b Inhibited the Expression of ASICs in the Acidic Microenvironment Conditions and Promoted the Expression of ECM-Related Genes and Proteins",
"section_num": null
},
{
"section_content": "To explore the mechanism by which Sa12b improves the biological activity of NP-MSCs cultured in an acidic environment, we used cells without Sa12b treatment as a control and obtained the optimal Sa12b concentration (8 μg/ μl) in the treatment group based on the results of proliferation and apoptosis experiments.As shown in Figure 6, the qPCR results showed that the expression of stem cell-related genes (Oct4, Nanog, Jagged, and Notch1) in the treatment group supplemented with Sa12b was significantly higher than that in the control group (p < 0.05) (Figure 6A).To measure Ca 2+ concentration, we treated NP-MSCs cultured in an acidic environment with the following: no Sa12b, Sa12b at an optimal concentration, amiloride (an ASIC non-specific inhibitor), and Sa12b and amiloride together.The results showed that the Ca 2+ concentration in the three treatment groups was lower than that in the control group.Interestingly, the Ca 2+ concentration in the group treated with Sa12b and amiloride simultaneously was significantly less than that in the control group (Figures 6B-D). ",
"section_name": "Sa12b Increased the Expression of Stem cell-Related Genes Under Acidic Microenvironmental Conditions and Reduced the Influx of Ca 2+ in NP-MSCs by Inhibiting ASICs",
"section_num": null
},
{
"section_content": "During the process of IDD, the change in the microenvironment, especially the low pH, is one of the key factors that inhibit the differentiation of NP-MSCs into NPlike cells (Kanichai et al., 2008;Wuertz et al., 2008;Wuertz et al., 2009;Li et al., 2013;Colombier et al., 2014), mainly by activating ASICs (Liu et al., 2017).In our study, we found that the newly emerged wasp peptide Sa12b (EDVDHVFLRF-), a non-specific ASIC inhibitor, could effectively improve the biological activity of human degenerated NP-MSCs in harsh acidic environments (pH = 6.2).Compared with that in the control, the proliferation rate of NP-MSCs increased with the increase in Sa12b concentration, and the apoptosis rate decreased.Moreover, the relative expression of genes encoding ASIC subunits (ASIC1, ASIC2, ASIC3, and ASIC4) expressed in human degenerated NP-MSCs affected by Sa12b was reduced.On the contrary, the expression of genes related to the ECM (collagen II, aggrecan, and SOX-9) and the protein expression of collagen II and aggrecan increased.Therefore, Sa12b improved the biological activity of human degenerated NP-MSCs and is very likely to contribute to remodeling the ECM of the NP tissue and delaying IDD.Furthermore, Sa12b inhibits Ca 2+ influx in human degenerated NP-MSCs by mediating ASICs to affect proliferation, differentiation, and apoptosis.In summary, our study indicated that the new ASIC non-specific inhibitor Sa12b could improve the biological activity of human degenerative NP-MSCs in a harsh acidic microenvironment and play a beneficial role in the process of IDD. The chronic decrease in the number and function of NPCs in NP under conditions of IDD and loss of ECM components, especially collagen II and aggrecan, is one of the pathological features of IDD (Antoniou et al., 1996;Roberts et al., 2006;Nicholas and George, 2011;Colombier et al., 2014;Risbud and Shapiro, 2014).Studies have found that NP tissue contains MSClike cells that can be isolated and proliferated in vitro, namely NP- MSCs, which have similar biological activity to bone marrow mesenchymal stem cells (BMSCs) and adipose-derived stem cells (ADSCs) (Blanco et al., 2010;Shen et al., 2015;Clouet et al., 2019), They can self-renew and differentiate into NPCs, promoting the regeneration of ECM after activation (Sakai et al., 2012).NP-MSCs were isolated from the NP in patients who underwent lumbar disc herniation surgery and then were extracted and purified.The homology observed in vivo and in vitro made this research highly relevant to clinical applications.In the cell identification stage, the cells presented the following characteristics: 1) adherent and long-term growth; 2) high expression of CD73, CD90, and CD105, and low expression of CD34, CD45, and HLA-DR; 3) osteogenic, adipogenic, and chondrogenic differentiation; and 4) expression of genes related to stem cells (Oct4, Nanog, Jagged, and Notch1), which met the international standards of MSCs and is consistent with the research of Shen et al. (Dominici et al., 2006;Shen et al., 2015). An acidic environment is vital for the progression of IVDD.ASICs, which are closely related to the acidic environment, affect the biological activity of cells by regulating the flow of ions inside and outside the cell (Zhou et al., 2016).Relevant studies have shown that acidic conditions can significantly inhibit the proliferation of human degenerated NP-MSCs and increase cell apoptosis through ASICs.As the pH of the culture environment of human degenerated NP-MSCs decreases, the expression of ASIC1, ASIC2, ASIC3, and ASIC4 gradually increases (Liu et al., 2017).In this study, we found that when the non-specific ASIC inhibitor Sa12b was not added to the culture, the cell proliferation rate decreased as the pH value decreased, and apoptosis increased, which was most apparent when the pH was 6.2 and is consistent with the study by Liu et al.After increasing Sa12b concentration, the proliferation increased, and the apoptosis rate decreased significantly, reaching the optimum level when the Sa12b concentration was 8 μg/μL qPCR showed that when the pH of the complete medium was 6.2 and different doses of Sa12b were added, the relative expression levels of ASIC1, ASIC2, ASIC3, and ASIC4 in NP-MSCs were all reduced.This illustrated that Sa12b affected human degenerative NP-MSCs through ASICs.In addition, studies have shown that endogenous stem cells such as NP-MSCs can self-renew and differentiate into NPCs, which can promote the regeneration of ECM after activation (Sakai et al., 2012).ECM receptor interaction can also activate the AKT pathway to promote the differentiation of neuronal stem cells (He et al., 2021).In addition, liver-specific ECM (L-ECM) can promote the liver differentiation of BM-MSCs by activating specific types of integrins (ITG) and its downstream signal ITG pathways, and play a therapeutically beneficial effect on the liver regeneration of stem cells (Bi et al., 2017).It is consistent with our results, as the concentration of Sa12b increased, the relative expression levels of ECM-related genes and proteins in NP-MSCs, such as collagen II, aggrecan, and SOX-9, were all increased; thus, we speculated that Sa12b had a high probability of promoting the regeneration of ECM in IVDD, promote the differentiation of NP-MSCs through downstream AKT and ITG, and delay IVDD. In addition, the Notch signaling pathway consists of four Notch receptors (Notch 1-4) and five Notch ligands (Jag1, Jag2, DLL-1, DLL-3, and DLL-4) (Siebel and Lendahl, 2017).The Notch signaling pathway, a key regulator of chondrogenesis, can regulate chondrocyte proliferation and differentiation and maintain the stromal metabolic balance of cartilage in articular chondrocytes (Liu et al., 2016).In addition, the study found that the binding of Notch1 and Jag1 activates the signal molecule HES1, and the activated HES1 inhibits the expression of downstream related genes, thereby inhibiting cell apoptosis and promoting cell cycle progression (Xue et al., 2014;Lee et al., 2016).This study found that the expression levels of Notch1 and Jag1 increased significantly when the Sa12b concentration was optimal (8 μg/μl).Moreover, the qPCR results indicated that the expression levels of each ASICs subunit decreased.Therefore, we believe that Sa12b may activate the Notch signaling pathway by mediating ASICs, increasing the expression of downstream genes in the pathway, promoting proliferation, and inhibiting the apoptosis of NP-MSCs. Oct4 and Nanog are homologous domain transcription factors that are the core transcription factors of human stem cells.Located upstream of genes responsible for totipotency regulation, Oct4 can maintain the pluripotency of stem cells (Saunders et al., 2017).Oct4 can be directly bound to the Nanog promoter to maintain the activation of Nanog and improve the proliferation ability of cells.Studies have found that upregulation of Oct4 and Nanog expression in dental pulp cells can promote cell proliferation, whereas downregulation can inhibit cell proliferation (Huang et al., 2014).In this study, the expression of Oct4 and Nanog was detected by qPCR, and the results showed that the expression was significantly increased at the optimal Sa12b concentration (8 μg/μl) compared with that in the control group.Therefore, Sa12b may promote the expression of Oct4 and then activate Nanog expression to enhance the pluripotency, proliferation, and differentiation of NP-MSCs. ASIC1 and ASIC3 are crucial subunits in the cytomembrane and are key mediators of human NP-MSC aging during IVDD.They are affected by changes in the levels of extracellular lactate and induce Ca 2+ influx.Ca 2+ , a key second messenger in the signal transduction pathway, induces the activation of the NF-κB signaling pathway, leading to apoptosis (Ding et al., 2021;Zhao et al., 2021).In vitro animal experiments have also shown that ASIC1 mediates cartilage endplate apoptosis and matrix metabolism under acidic conditions (Yuan et al., 2016).In addition, the reduction in the pH of the articular fluid can lead to excessive apoptosis of chondrocytes.Wu et al. found that ASIC1a may promote [Ca 2+ ] i and upregulate NLRP3 inflammasome expression, thus mediating cocaine death in AA rat chondrocytes (Wu et al., 2019). Our results showed that the Ca 2+ influx in the three treatment groups was lower than that in the control group.Interestingly, the Ca 2+ influx in the group treated with Sa12b and amiloride simultaneously was significantly less than that in the control group.Studies have shown that in articular cartilage tissue and IVD, when the pH is within the physiological range, Ca 2+ has a very high affinity for ASICs and can bind to the outside of the channel holes to close the channels; when pH decreases, H+ binds to acid-sensitive sites on the ASICs, reducing the binding affinity of Ca 2+ to the ASICs, and Ca 2+ leaves the channel pore, promoting the opening of the ASICs and entering the cell (Zhang et al., 2020).In addition, scientists speculated that the inhibitory effect of Sa12b on ASICs may occur because Sa12b has two positively charged residues (His5 and Arg9) that can bind to the binding site of the ASIC, especially the central vestibule of the channel, thereby blocking the channel (Hernández et al., 2019).Moreover, related studies have shown that ASIC1 and ASIC3 activate the senescence programming pathway of P53-P21/P27 and P16-RB1 signaling in an acid environment, and the expression of genes P53, P21, P27, P16 and RB1 increases, which promotes human degenerative disc NP-MSCs senescence and degeneration (Ding et al., 2021).The acid environment also regulates the level of reactive oxygen species between NP cells differentiated from human degenerated intervertebral disc NP-MSCs through ASIC1 and ASIC3, activates the NF-κB signaling pathway, and promotes intracellular activation of the NLRP3 inflammation group and IL-1β release, thereby promoting NP degeneration (Zhao et al., 2021).Therefore, we believe that Sa12b is very likely to reduce the Ca 2+ influx by inhibiting AISCs, reduce the expression of genes related to the aging programming pathway of P53-P21/P27 and P16-RB1 signal transduction, and improve the biological activity of human degenerated intervertebral disc NP-MSCs.And inhibit or avoid activation of NF-κB signal to reduce the expression of inflammatory factors, thereby delaying IVDD. Our study demonstrated that in the harsh acidic environment (pH = 6.2), the inhibition of AISCs reduced the influx of Ca 2+ , thus improving the biological activities of NP-MSCs, such as proliferation, differentiation, and apoptosis.However, there were still some limitations in our study.First, we only studied the influence of Sa12b on the biological activity of NP-MSCs in a harsh acid environment, so further research on other IVD environments at different pH levels should be conducted.Second, although Sa12b reduces Ca 2+ influx by inhibiting AISCs, thereby mediating the improvement of the biological activity of NP-MSCs, the specific signal pathways through which Sa12b affects NP-MSCs need further study.Third, we only carried out in vitro studies on the biological activity of NP-MSCs in the model of an acidic environment.We did not investigate the effects of various factors in vivo.Therefore, animal experiments should be conducted in future studies. In summary, our study indicated that Sa12b reduces Ca 2+ influx by inhibiting AISCs in the harsh acidic environment of IVD and may improve the biological activity of NP-MSCs through the Notch signaling pathway.This study provides a new perspective for the biological treatment of IVDD.Sa12b has enormous therapeutic potential for delaying IVDD and improving low back pain. ",
"section_name": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "Thanks to Professor Meijuan Zheng's research team for supporting this study, providing flow cytometry, and Siyu Liu for teaching us how to analyze flow cytometry data analysis. ",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
},
{
"section_content": "This study was supported by the Natural Science Foundation of China (No.82072492). ",
"section_name": "FUNDING",
"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 authors. ",
"section_name": "DATA AVAILABILITY STATEMENT",
"section_num": null
},
{
"section_content": "The studies involving human participants were reviewed and approved by The First Affiliated Hospital of Anhui Medical University.The patients/participants provided their written informed consent to participate in this study.Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. HT and JL was responsible for the conception, study design, and final decision of the manuscript.ZW was responsible for the study design, data collection and analysis, and manuscript writing and revision.LH, SuZ, and ShZ contributed to the experimental studies and data collection.HC, HZ, and YL were responsible for the data analysis. 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 Wang, Han, Chen, Zhang, Zhang, Zhang, Li, Tao and Li.This is an open-access 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 studies involving human participants were reviewed and approved by The First Affiliated Hospital of Anhui Medical University.The patients/participants provided their written informed consent to participate in this study.Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. ",
"section_name": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "HT and JL was responsible for the conception, study design, and final decision of the manuscript.ZW was responsible for the study design, data collection and analysis, and manuscript writing and revision.LH, SuZ, and ShZ contributed to the experimental studies and data collection.HC, HZ, and YL were responsible for the data analysis. ",
"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. Copyright © 2022 Wang, Han, Chen, Zhang, Zhang, Zhang, Li, Tao and Li.This is an open-access 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": "Conflict of Interest:",
"section_num": null
}
] |
10.1186/bcr2347
|
The cytotoxicity of γ-secretase inhibitor I to breast cancer cells is mediated by proteasome inhibition, not by γ-secretase inhibition
|
<jats:title>Abstract</jats:title><jats:sec> <jats:title>Introduction</jats:title> <jats:p>Notch is a family of transmembrane protein receptors whose activation requires proteolytic cleavage by γ-secretase. Since aberrant Notch signaling can induce mammary carcinomas in transgenic mice and high expression levels of Notch receptors and ligands correlates with overall poor clinical outcomes, inhibiting γ-secretase with small molecules may be a promising approach for breast cancer treatment. Consistent with this hypothesis, two recent papers reported that γ-secretase inhibitor I (GSI I), Z-LLNle-CHO, is toxic to breast cancer cells both in vitro and in vivo. In this study, we compared the activity and cytotoxicity of Z-LLNle-CHO to that of two highly specific GSIs, DAPT and L-685,458 and three structurally unrelated proteasome inhibitors, MG132, lactacystin, and bortezomib in order to study the mechanism underlying the cytotoxicity of Z-LLNle-CHO in breast cancer cells.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>Three estrogen receptor (ER) positive cell lines, MCF-7, BT474, and T47D, and three ER negative cell lines, SKBR3, MDA-MB-231, and MDA-MB-468, were used in this study. Both SKBR3 and BT474 cells also overexpress HER2/neu. Cytotoxicity was measured by using an MTS cell viability/proliferation assay. Inhibition of γ-secretase activity was measured by both immunoblotting and immunofluorescent microscopy in order to detect active Notch1 intracellular domain. Proteasome inhibition was determined by using a cell-based proteasome activity assay kit, by immunoblotting to detect accumulation of polyubiquitylated protein, and by immunofluorescent microscopy to detect redistribution of cellular ubiquitin.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>We found that blocking γ-secretase activity by DAPT and L-685,458 had no effect on the survival and proliferation of a panel of six breast cancer cell lines while Z-LLNle-CHO could cause cell death even at concentrations that inhibited γ-secretase activity less efficiently. Furthermore, we observed that Z-LLNle-CHO could inhibit proteasome activity and the relative cellular sensitivity of these six breast cancer cell lines to Z-LLNle-CHO was the same as observed for three proteasome inhibitors. Finally, we found that the cell killing effect of Z-LLNle-CHO could be reversed by a chemical that restored the proteasome activity.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>We conclude that the cytotoxicity of Z-LLNle-CHO in breast cancer cells is mediated by proteasome inhibition, not by γ-secretase inhibition.</jats:p> </jats:sec>
|
[
{
"section_content": "Notch is a family of single-pass type I transmembrane protein receptors that, in mammals, includes four homologs, Notch1 to 4 [1].Ligand-induced Notch receptor activation requires at least two cleavages that release the intracellular domain from the cytomembrane and allow it to translocate into the nucleus where it activates its target genes [1].The final cleavage is performed by γ-secretase, whose substrates include all four Notch receptors and their ligands as well as β-amyloid precursor protein, E-cadherin, CD44, ErbB-4, and ephrin-B1 [2][3][4][5][6][7][8]. Aberrant Notch signaling can induce oncogenesis and may promote the progression of breast cancer.Transgenic mice overexpressing active Notch1, Notch3, or Notch4 homologs all developed mammary carcinoma [9,10].Furthermore, a recent clinical study reported that the expression level of DMEM: Dulbecco's modified eagle's medium; DMSO: dimethyl sulfoxide; ER: estrogen receptor; FBS: fetal bovine serum; GSI: γ-secretase inhibitor; N1ICD: Notch1 intracellular domain; N1EXT: Notch1 extracellular truncation; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; SD: standard deviation. Notch1, Notch3, and JAG-1, one of the Notch ligands, were inversely correlated with the overall clinical outcomes in breast cancer patients [11].These observations have prompted great interest in targeting Notch signaling in breast cancer for therapeutic benefit.However, it should be noted that Notch2 signaling has been reported to function as a tumor suppressor in breast cancer cells [12]. Among the several options to block Notch signaling, inhibition of γ-secretase by small molecules offers a promising approach and has been used extensively to study the downstream targets of the Notch signaling pathway [13,14].However, experimental data supporting the concept that γ-secretase inhibitors (GSIs) could inhibit the growth of, or kill, breast cancer cells have been scarce.Two recent reports have provided the strongest evidence by showing that Z-LLNle-CHO, commonly considered to be a GSI, has such an effect both in vitro and in vivo [15,16]. Proteasome inhibitors are a class of recent developed anticancer drugs.Z-LLNle-CHO, as a derivative of a widely used proteasome inhibitor MG-132, has been reported to inhibit chymotryptic protease activity, a core function of the proteasome [17].In this study, we compared the activity and cytotoxic effects of Z-LLNle-CHO with those of two other widely used and highly specific GSIs, DAPT and L-685,458, and with those of three structurally unrelated proteasome inhibitors, MG132, lactacystin, and bortezomib.Our results suggest that the cell killing effect of Z-LLNle-CHO is not mediated by γsecretase inhibition, but is mediated by proteasome inhibition. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "Z-Leu-Leu-Nle-CHO (Z-LLNle-CHO, also called GSI I), N-(N-(3,5-Difluorophenacetyl-L-alanyl))-S-phenylglycine t-Butyl Ester (commonly called DAPT or GSI IX), (1S-Benzyl-4R-(1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl)-2R-hydroxy-5-phenylpentyl) carbamic acid tert-butyl ester (commonly called L-685,458 or GSI X), Z-Leu-Leu-Leualdehyde (Z-LLL-CHO, commonly referred to as MG132), lactacystin, and edaravone were purchased from Calbiochem (San Diego, CA, USA) and dissolved in dimethyl sulfoxide (DMSO).Bortezomib was purchased from LC Laboratories (Woburn, MA, USA) and dissolved in DMSO.Tiron was from Sigma (St. Louis, MO, USA) and dissolved in water. ",
"section_name": "Reagents",
"section_num": null
},
{
"section_content": "Three estrogen receptor (ER) positive cell lines, MCF-7, T47D, and BT474, and three ER negative cell lines, SKBR3, MDA-MB-231, and MDA-MB-468, were used in this study.Both SKBR3 and BT474 cells also overexpress HER2/neu.The culture medium was DMEM/F-12 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco) and GlutaMAX (Gibco) for all cell lines except SKBR3, which was cultured in McCoy's 5A medium (Gibco) supplemented with 10% FBS and GlutaMAX.In addition, MCF-7 culture medium was supplemented with non-essential amino acids (Gibco), sodium pyruvate (Gibco)and 10 μg/ml of insulin (Sigma).T47D culture medium was also supplemented with insulin (10 μg/ml).All cell lines were maintained at 37°C in a humidified atmosphere of 5% carbon dioxide in air. ",
"section_name": "Cell culture",
"section_num": null
},
{
"section_content": "Cell viability and proliferation was measured using the Cell-Titer 96 ® AQ ueous One Solution Cell Proliferation Assay (MTS) kit (Promega, Madison, WI, USA).Cells (3000 to 8000 cells/ well) were seeded into 96-well plates in triplicate and allowed to attach overnight before being treated with increasing concentrations of the drugs.All wells, including the control, were exposed to the same concentration of DMSO to eliminate any possible effect of the vehicle on cell viability and proliferation.MTS reagent (20 μl) was added to each well 72 hours later and, after one to four hours incubation, the absorbance at 490 nm was measured using a microplate reader (FLUOstar OPTIMA from BMG LABTECH, Offenburg, Germany).Relative cell viability and proliferation of individual samples was calculated by normalizing their absorbance to that of the corresponding control sample.The mean and standard deviation (SD) of three independent experiments were used to plot dose-response curves.The concentrations that kill and/or inhibit cell growth by 50% (EC 50 ) were calculated from the equations that best fit the linear range of the dose-response curves. ",
"section_name": "Cell viability and proliferation assay",
"section_num": null
},
{
"section_content": "Cells at 80% confluence were treated overnight with drugs at the indicated concentrations and control cultures received DMSO.The next day, cells were incubated with trypsin/EDTA (Gibco) solution for 10 minutes before collection by centrifugation.Cell pellets were then washed once with ice-cold PBS, lysed in lysis buffer (100 mM Tris-HCl (pH 6.8), 10% glycerol, 2% SDS, 1 mM EDTA, 0.002% bromophenol blue, 2 mM NaF, 1 mM Na 3 VO 4 , 1 × protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA)), boiled for five minutes, and passed through a 21 gauge needle.The positive control samples were prepared in the same way as the GSI-treated samples and the negative control samples were prepared by adding the lysis buffer directly to the culture plates after washing with PBS without trypsin/EDTA incubation.Protein concentrations were quantified using a BCA protein assay (Pierce, Rockland, IL, USA). ",
"section_name": "Protein sample preparation",
"section_num": null
},
{
"section_content": "Protein samples (50 μg/lane) were separated in 8% SDS-PAGE gels and transferred to Trans-Blot ® pure nitrocellulose membranes (0.2 μm, Bio-Rad, Hercules, CA, USA).The membranes were blocked with 5% skim milk in TTBS (0.1% Tween-20, 100 mM Tris-HCl (pH 7.4), 150 mM NaCl) at room temperature for one hour before being probed overnight at 4°C with primary antibody solution.The primary antibodies used were anti-Notch1 (Val1744; Cell Signaling Technology, Danvers, MA, USA, 1:1000), anti-ubiquitin (clone FK2 from Millipore, Billerica, MA, USA, 1:1000) and anti-actin (Abcam, Cambridge, MA, USA, 1:5000).After washing with TTBS four times for 10 minutes each, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or antimouse (Jackson ImmunoResearch Laboratories, West Grove, PA, USA, 1:15,000) secondary antibody solution at 4°C for three hours.After another round of four washes with TTBS, the membranes were incubated with SuperSignal West Pico Chemiluminescent Substrate (Pierce), exposed to Fuji (Tokyo, Japan) film, and then developed to visualize the protein signal. ",
"section_name": "Western blot analysis",
"section_num": null
},
{
"section_content": "Synthetic DNA oligonucleotides corresponding to the cDNA encoding human Notch1 signal peptide flanked by restriction enzyme recognition sequences were integrated into pCMV-Tag4A vector (Stratagene, La Jolla, CA, USA) using Sac II/ BamH I sites.Then the cDNA encoding the amino acid residues 1721 to 2555 (corresponding to the substrate of γsecretase) was amplified using reverse transcription-coupled PCR of MCF-7 total cellular RNA and integrated into the vector containing the Notch1 signal peptide-encoding sequence using BamH I/EcoR I sites.The sequence of the new construct was verified by sequencing using T3/T7 primers. ",
"section_name": "Construction of flag-tagged Notch1 extracellular truncation (N1EXT) vector",
"section_num": null
},
{
"section_content": "N1EXT plasmid DNA was transfected into MCF-7 and SKBR3 cells plated on glass coverslips using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA).Culture medium was replaced six hours after transfection with fresh medium containing 5 μM of DAPT, 2 μM of L-685,458, or Z-LLNle-CHO at the calculated EC 50 values of individual cell lines.After overnight incubation to allow the expression of exogenous protein, cells were fixed with 4% paraformaldehyde solution for indirect immunofluorescent microscopy. ",
"section_name": "Transfection and treatment",
"section_num": null
},
{
"section_content": "Fixed cells were first permeabilized with 0.5% Triton X-100 in PBS at room temperature for five minutes and then probed with anti-Flag monoclonal antibody (clone M2 from Sigma, 1:500) at room temperature for one hour.After five washes with PBS, cells were incubated with Alexa 488-conjugated goat anti-mouse secondary antibody (Molecular Probes, Carlsbad, CA, USA, 1:250) at room temperature for 45 minutes and further counterstained with 0.5 μg/ml of DAPI after five washes with PBS.Images were taken using LSM 510 laser scanning confocal microscope with a Plan-Neofluar 40X/ 1.3NA oil-immersion objective lens (Carl Zeiss, Jena, Germany).The optical slice thickness was less than 0.9 μm. ",
"section_name": "Indirect immunofluorescent microscopy",
"section_num": null
},
{
"section_content": "MCF-7 and MDA-MB-231 cells plated on glass coverslips were treated with drugs at the indicated concentrations for four hours before being fixed in 4% paraformaldehyde solution.Fixed cells were immunostained in the same way as above except that anti-ubiquitin monoclonal antibody (clone FK2 from Millipore, 1:1,000) was used as the primary antibody.Images were taken using LSM 710 laser scanning confocal microscope with a Plan-Apochromat 20X/0.8NAobjective lens (Carl Zeiss).The optical slice thickness was 1.8 μm. ",
"section_name": "Determination of ubiquitin distribution",
"section_num": null
},
{
"section_content": "Proteasome activity was measured using the Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay kit (Promega, Madison, WI, USA).Briefly, MCF-7 (6000 cells/well) and MDA-MB-231 (10 4 cells/well) cells were plated into whitewalled 96-well plates.After overnight incubation to allow cell attachment, cells were treated with drugs at indicated concentrations for two hours.Equal volumes of Proteasome-Glo™ reagent were then added and the luminescence signal was measured using a microplate reader (FLUOstar OPTIMA). ",
"section_name": "Proteasome activity assay",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "We first compared the cytotoxicity of Z-LLNle-CHO to two other widely used GSIs, DAPT and L-685,458.Treatment with Z-LLNle-CHO resulted in a dose-dependent decrease in cell viability/proliferation of all six breast cancer cell lines tested with ER-negative cell lines being more sensitive than ER-positive cell lines.The calculated ED 50 values were 3.25 μM, 2.5 μM, 2.4 μM, 1.8 μM, 1.6 μM, and 1.4 μM for MCF-7, BT474, T47D, MDA-MB-231, SKBR3, and MDA-MB-468, respectively.However, DAPT and L-685,458 had no cell killing and/ or growth inhibitory effects at concentrations up to 5 μM and 2 μM, respectively (Figure 1). ",
"section_name": "Among the three GSIs, only Z-LLNle-CHO induces cell death",
"section_num": null
},
{
"section_content": "We then examined whether the lack of cell killing/growth inhibition by DAPT and L-685,458 was due to their lower potency in inhibiting γ-secretase activity.To address this question, we first performed immunoblot analysis using an antibody that only recognizes cleaved Notch1 intracellular domain (N1ICD) [18,19].As N1ICD is a product of γ-secretase, its abundance is a good indicator of γ-secretase activity.However, the endogenous N1ICD level (the negative control lanes in Figure 2a) is too low to be detected confidently.Therefore, we took advantage of the fact that calcium depletion activates Notch1 in the absence of ligand binding [20].As shown in Figure 2a, DAPT at 5 μM and L-685,458 at 2 μM could block calcium depletion-induced Notch1 cleavage in all six cell lines.At the same time, Z-LLNle-CHO, at the concentrations that inhibited cell growth/viability by 50%, failed to do so to a comparable level in SKBR3 and MDA-MB-468 cells, although similar inhibition was observed in the other four cell lines treated with this drug. To confirm the potency of DAPT and L-685,458 on inhibiting γ-secretase activity in intact cells, we transfected MCF-7 and SKBR3 cells with a plasmid expressing a Flag-tagged N1EXT fragment that mimics the immediate substrate of γ-secretase and then treated them with the same concentrations of GSIs as used for the western blot analysis.Without any intervention, the exogenous protein will be cleaved by γ-secretase as long as it is transported to the plasma membrane to produce N1ICD that can be visualized as nuclear signal when transfected cells are immunostained with an anti-Flag antibody (control panels in Figure 2b).In contrast, when γ-secretase activity is inhibited, the exogenous protein cannot be cleaved and therefore will accumulate at the plasma membrane.As shown in Figure 2b, all the DAPT-and L-685,458-treated cells and Z-LLNle-CHO-treated MCF-7 cells showed exclusively membrane signal.However, 24% and 58% of Z-LLNle-CHOtreated SKBR3 cells displayed mainly nuclear signal or a mixture of nuclear and plasma membrane signal, respectively.This is consistent with the immunoblotting data demonstrating that DAPT and L-685,458 could completely inhibit γ-secretase activity at tested concentrations in both cell lines but Z-LLNle-CHO could only do so in MCF-7 cells (Figure 2c). Taken together, because complete inhibition of γ-secretase activity by two structurally unrelated GSIs had no effect on cell viability and proliferation, it is unlikely that the cell killing/ growth inhibitory effect of Z-LLNle-CHO on breast cancer cell lines was mediated by γ-secretase inhibition. ",
"section_name": "All three GSIs inhibit γ-secretase activity",
"section_num": null
},
{
"section_content": "Z-LLNle-CHO is derived from a widely used proteasome inhibitor MG132 (Z-LLL-CHO) and has been reported to be a broad chymotryptic and aspartyl protease inhibitor [17].Therefore, we examined whether Z-LLNle-CHO also has proteasome inhibitor activity at the concentrations that showed dose-dependent cytotoxicity.We first used a cell-based proteasome activity kit to measure proteasome activity after cells were treated with MG132, Z-LLNle-CHO, or DAPT.As shown in Figure 3b, both Z-LLNle-CHO and MG132 showed a dosedependent inhibition of the proteasome at concentrations that showed cytotoxic effects, although DAPT did not.Next, we examined whether or not inhibition of proteasome activity caused accumulation of polyubiquitinated protein, one of the major causes of proteasome inhibitor-induced cell death [21], by subjecting the protein samples from cells treated with 5 μM (MCF-7) or 2.5 μM (MDA-MB-231) of Z-LLNle-CHO overnight to immunoblotting with an anti-ubiquitin antibody.We used bortezomib, a specific proteasome inhibitor that has been approved to treat multiple myeloma in patients, as the positive control.The results showed that treatment with Z-LLNle-CHO indeed resulted in the same accumulation of polyubiquitinated protein that was observed with bortezomib (lane 2 and 5 of Figure 3b).Finally, we took advantage of a recent observation that when proteasome-mediated protein degradation was inhibited, cellular ubiquitin would undergo a nuclear-to-cytoplasmic redistribution that could be detected by anti-ubiquitin FK2 antibody [22].In untreated MCF-7 and MDA-MB-231 cells, FK2 staining showed dominant nuclear signal (Figure 3c).After a four hour treatment with either bortezomib or Z-LLNle-CHO but not with DAPT, cells displayed a strong cytoplasmic ubiquitin signal, confirming proteasome activity was inhibited by Z-LLNle-CHO. ",
"section_name": "Z-LLNle-CHO has proteasome inhibitory activity",
"section_num": null
},
{
"section_content": "We next asked whether or not the cell killing effect of Z-LLNle-CHO is mediated by its proteasome inhibition activity.If this is The redistribution of nuclear ubiquitin to cytoplasm is a phenomenon that can be induced by proteasome inhibition. number not for citation purposes) the case, the relative cellular sensitivity of different breast cancer cell lines to Z-LLNle-CHO should reflect that produced by other proteasome inhibitors.Therefore, we treated the same six breast cancer cell lines with increasing doses of three structurally unrelated proteasome inhibitors, MG132, lactacystin, and bortezomib, and measured the effects on cell viability/proliferation using the MTS assay.Similar to the results shown in Figure 1, ER-positive cell lines were more resistant to all the three proteasome inhibitors than ER-negative cell lines (Figure 4).In addition, our results were also consistent with a previous study using bortezomib alone [23].These data strongly suggest that the cell killing effects of Z-LLNle-CHO in breast cancer cells is mediated by its proteasome inhibitory function. ",
"section_name": "The cellular sensitivity of six breast cancer cell lines to Z-LLNle-CHO is the same as that to proteasome inhibitors",
"section_num": null
},
{
"section_content": "Recent studies showed that the proteasome inhibitory activity as well as the cell killing effects of bortezomib and MG132 could be specifically blocked by two antioxidants, tiron and edaravone, respectively [24,25].As Z-LLNle-CHO is structurally similar to MG132, we speculated that edaravone might also be able to reverse the cytotoxicity of Z-LLNle-CHO by blocking its proteasome inhibition activity.Therefore, we first treated MCF-7 and MDA-MB-231 cells with different combinations of bortezomib or Z-LLNle-CHO and tiron or edaravone, and then measured cell growth using the MTS assay.Consistent with previous studies, tiron but not edaravone rescued cells from bortezomib-induced cell killing.Most importantly, we found edaravone but not tiron could rescue cells from Z-LLNle-CHO-induced cell killing (Figure 5a). Next, we tested whether or not edaravone could rescue proteasome activity from Z-LLNle-CHO-induced inhibition.We exposed cells to edaravone at the concentration that showed best cell growth rescue in the presence of Z-LLNle-CHO and measured proteasome activity using the three approaches we used above.We used tiron to reverse bortezomib-induced proteasome inhibition as a control.We found that edaravone indeed rescued the proteasome activity from Z-LLNle-CHOinduced, but not bortezomib-induced, inhibition.Although the proteasome activity of edaravone rescued from Z-LLNle-CHOinduced inhibition was not to the same extent as tiron rescued bortezomib-induced inhibition in the cell based proteasome assay (Figure 5b), the rescued proteasome activity was enough to prevent the accumulation of polyubiquitinated proteins (lane 4 compared with lane 2 in Figure 3b) and redistribution of cellular ubiquitin (Figure 3c, treatment 4 vs. treatment 2).In addition, we found edaravone also partially restored γsecretase activity from Z-LLNle-CHO-induced inhibition (Figure 5c). ",
"section_name": "The cytotoxicity of Z-LLNle-CHO can be reversed by a specific antioxidant that restores proteasome activity",
"section_num": null
},
{
"section_content": "To investigate whether the cytotoxicity of Z-LLNle-CHO to breast cancer cells is due to the summation or synergy of its dual activities, we tested whether a combination of a specific γ-secretase inhibitor with a specific proteasome inhibitor could produce an additive or synergetic effect on cell killing.We subjected cells to increasing concentrations of lactacystin with or without 5 μM of DAPT that completely inhibited γsecretase activity in the cell lines tested.We found the doseresponse curves of individual cell lines treated with or without DAPT was almost identical (Figure 6), which suggests there was no additive or synergetic effects of inhibiting both γ-secretase activity and proteasome activity.Therefore, γ-secretase inhibitory activity of Z-LLNle-CHO most likely does not contribute to its cell killing effect in breast cancer cells. ",
"section_name": "γ-secretase inhibition activity of Z-LLNle-CHO does not contribute to its cytotoxicity to breast cancer cells",
"section_num": null
},
{
"section_content": "Blocking Notch signaling by inhibiting γ-secretase activity with small molecules has been suggested to be a promising The relative sensitivity of six cell lines to three proteasome inhibitors is the same as that to Z-LLNle-CHO The relative sensitivity of six cell lines to three proteasome inhibitors is the same as that to Z-LLNle-CHO.Cells were treated with MG132, bortezomib, or lactacystin at indicated concentrations for 72 hours before cell viability was measured by MTS assay.Results represent the mean ± standard deviation of three independent experiments.Please note the relative cellular sensitivity of the same six breast cancer cell lines to three structurally unrelated proteasome inhibitors was the same as that to Z-LLNle-CHO in Figure 1. approach to battle breast cancer [13,14].In fact, there are three ongoing clinical trials registered at ClinicalTrials.gov using GSIs in the treatment of breast cancer.However, experimental data supporting the effectiveness of GSIs in the inhibition of cell growth or killing of breast cancer cells have been scarce.Two recent reports, however, have now shown that Z-LLNle-CHO, commonly called GSI I, has such an effect both in vitro and in vivo [15,16]. In the present study, we first compared the cytotoxicity and activity of Z-LLNle-CHO with two other popularly used GSIs, DAPT and L-685,458.We found that completely inhibiting γ- The cytotoxicity effect of Z-LLNle-CHO could be reversed by edaravone that blocks its proteasome inhibitory function The cytotoxicity effect of Z-LLNle-CHO could be reversed by edaravone that blocks its proteasome inhibitory function.(a) Cells were treated with indicated drugs for 72 hours before cell growth was measured using MTS assay.Results represent the mean ± standard deviation of independent experiments.(b) Proteasome activity in intact cells was directly measured using a cell-based assay after cells were treated with different combinations of drugs for two hours.The treatment conditions were (from left to right): dimethyl sulfoxide (DMSO) vehicle only; bortezomib alone; bortezomib plus tiron; bortezomib plus edaravone; Z-LLNle-CHO alone; Z-LLNle-CHO plus tiron; and Z-LLNle-CHO plus edaravone.The concentrations of bortezomib, tiron, edavarone, and Z-LLNle-CHO are 100 nM, 2 mM, 100 μM, and 5 μM for MCF-7 cells, and 40 nM, 0.5 mM, 100 μM, and 2.5 μM for MDA-MB-231 cells, respectively.Results represent the mean ± SD of three independent experiments.(c) The same protein samples used for immunoblotting in Figure 3b plus another negative control sample were subjected to immunoblotting with anti-Notch1 (V1744) antibody that specifically recognizes active Notch1 intracellular domain.The order of the samples were the same as that in Figure 3b except that lane 1 is the new negative control sample. number not for citation purposes) secretase activity by DAPT and L-685,458 had no effect on cell viability and proliferation of a panel of six breast cancer cell lines with different genetic backgrounds.In contrast, Z-LLNle-CHO could cause cell death even at concentrations that did not completely inhibit γ-secretase activity.Therefore, we conclude that the cell killing effect of Z-LLNle-CHO on breast cancer cells is not mediated by γ-secretase inhibition. We next measured the proteasome inhibition potential of Z-LLNle-CHO.In contrast to two previous reports that Z-LLNle-CHO at concentrations that inhibited cell growth did not significantly inhibit proteasome activity (see supplemental materials in [15,26]), we found that it could inhibit proteasome activity by about 50% in intact cells even at a concentration that did not show significant cytotoxicity in two cell lines tested.Our result is consistent with a recent study that was published during the revision of this manuscript [27].The new study showed that Z-LLNle-CHO at about 0.3 μM (calculated by us based on scale) inhibited proteasome activity by 80% and slowed cell growth by 20% in MCF-7 cells.As the approach the new study used to measure proteasome activity is different from ours, the extent of proteasome activity inhibition cannot be compared between their data and ours.However, both studies show that Z-LLNle-CHO could significantly inhibit proteasome activity at concentrations that showed dose-dependent cytotoxicity.The previous two studies used the same method to measure proteasome activity as the latest study but differed from ours.Therefore, it is easy to explain the discrepancy between their data and ours but we cannot explain the discrepancy between their data and the latest study. Furthermore, we found that the relative cellular sensitivity of six breast cancer cell lines to Z-LLNle-CHO was the same as that to three widely used but structurally unrelated proteasome inhibitors and was also consistent with a previous study [23].This consistency strongly suggests that the cell killing effect of Z-LLNle-CHO is due to its proteasome inhibitory function.Most convincingly, we found that the cytotoxic effect of Z-LLNle-CHO could be reversed by a specific antioxidant that blocked its proteasome inhibitory activity.Finally, we tested but did not find any additive effect of the combination of a specific γ-secretase inhibitor and a specific proteasome inhibitor on breast cancer cell growth.Therefore, we conclude that the cytotoxicity of Z-LLNle-CHO to breast cancer cells is mediated by proteasome inhibition. We noticed that edaravone treatment also partially rescued γsecretase activity from Z-LLNle-CHO-induced inhibition.However, because inhibition of γ-secretase alone or in combination with proteasome inhibition had no effect on cell survival/proliferation or cellular response to proteasome inhibition, we do not consider partially restored γ-secretase activity as a major contributor to the reversion of the cytotoxicity induced by Z-LLNle-CHO.Likewise, although edaravone has been reported to protect cells from apoptosis by acting as an antioxidant [28,29], we do not think its free radical scavenging activity is a major contributor because it had no effect on bortezomibinduced cytotoxicity.Therefore, its ability to restore proteasome activity through unknown mechanism(s) most likely accounts for the reversion of the cytotoxicity of Z-LLNle-CHO. Both previous studies used transient transfection of N1ICD to rescue the cell death induced by Z-LLNle-CHO treatment and argued that the reversion of the phenotype by N1ICD transfection indicated that Z-LLNle-CHO induced cell death through inhibiting Notch signaling pathway [15,16].However, transient overexpression of N1ICD has been reported to inhibit wild-type p53-induced apoptosis in immortalized epidermal cells [30], to inhibit dexamethasone, etoposide, or Fas-ligandinduced apotosis in mature T-cells [31], and to protect H460 (lung cancer), HepG2 (liver cancer), and HT1080 (fibrosarcoma) from several chemotherapy drugs [32].Therefore, an alternative interpretation of the data from the two previous studies is that N1ICD over-expression provided pro-survival signals that antagonize the pro-apoptotic effects of Z-LLNle-CHO. It is worthy noting that many of the effects of Z-LLNle-CHO reported in previous studies, including G2/M arrest and regulation of apoptosis-related protein, are consistent with the reported effects of other proteasome inhibitors [33][34][35][36][37].In addition, similar to the additive effects of 4-OH-TAM and Z-LLNle-CHO on the inhibition of T47D:A18 cells growth [15], additive or even synergistic effects have also been reported between tamoxifen and bortezomib in some but not all ER-positive breast cancer cell lines tested [38,39].Although the sim- ilarities between the biological effects of Z-LLNle-CHO and those of other proteasome inhibitors do not necessarily mean that they function the same, our finding that Z-LLNle-CHO inhibits breast cancer cell growth as a proteasome inhibitor can explain the data produced with the use of Z-LLNle-CHO in previous studies. It should be pointed out that although the latest study by Rasul and colleagues found that Z-LLNle-CHO has proteasome inhibitory function at concentrations that showed dosedependent cytotoxicity [27], the authors did not consider its proteasome inhibitory activity as the major contributor to its cell killing effects because the cytotoxicity of Z-LLNle-CHO and MG132 was 'markedly different', although their proteasome inhibition potential was similar.However, by careful analysis of their data, we found that the proteasome inhibition potentials of Z-LLNle-CHO and MG132 differed by more than two-fold, not less than the difference in cytotoxicity, within the range of concentrations that Z-LLNle-CHO and MG132 showed 'markedly different' cytotoxicity (below 0.6 μM).Most importantly, Z-LLNle-CHO at 0.75 μM in their study slowed MCF-7 cell growth by 80%, but only inhibited γ-secretase activity by 25%.Meanwhile, it inhibited proteasome activity by 80%.Therefore, their data is more consistent with our conclusion that the cytotoxicity of Z-LLNle-CHO was not due to γsecretase inhibition, but due to proteasome inhibition. The observation that both Z-LLNle-CHO and MG132 at given concentrations inhibited proteasome activities to comparable levels in MCF-7 and MDA-MB-231 cells, but showed different cytotoxicity, is not surprising because this has also been observed for bortezomib [23].The reduced sensitivity of ERpositive MCF-7 cells may be a consequence of pro-survival signal provided by the ER signaling pathway in these ER-positive breast cancer cells.This hypothesis is consistent with the observed additive or even synergistic effect between tamoxifen and Z-LLNle-CHO or bortezomib.However, this requires further investigation.Regardless of the mechanisms, our results, together with the previous reports, suggest that the future clinical trials testing the effectiveness of proteasome inhibitors in treating breast cancer should take the ER status into consideration when enrolling patients. The observation that two specific GSIs, DAPT and L-685,458, had no effect on the survival and proliferation of breast cancer cells does not eliminate the potential use of GSIs or other approaches to block Notch signaling for breast cancer treatment.The results presented here were obtained from in vitro cell culture experiments.The effects of GSIs on the tumors grown in vivo, where the Notch signaling might be more active due to enhanced ligand-receptor interaction, could be different and need further investigation.Alternatively, these drugs might block the signaling pathway of some as yet unidentified substrate(s) which antagonizes the effect of reduced Notch1 signaling on breast cancer cell survival and proliferation.There are at least a dozen known γ-secretase substrates and most of the available GSIs have no preference for specific substrates.Rather than laboriously testing all potential candidates that antagonize Notch1, it might be better to develop substratespecific GSIs.To this end, it is encouraging to note that compounds that can preferentially modulate γ-secretase activity against Aβ42 over Notch have recently been reported [40].These compounds target the substrate (Aβ42) rather than the γ-secretase active site itself.In principle, it should also be possible to find drugs that target individual Notch homologs.Alternatively, it might be useful to develop neutralizing antibody against individual Notch homologs just as the trastuzumab targets HER2/neu.Furthermore, the results of this study do not diminish the potential use of Z-LLNle-CHO for breast cancer treatment.In fact, we believe that clarifying its role as a proteasome and γsecretase dual inhibitor will help to direct its potential development for clinical use.However, we do caution that results obtained using Z-LLNle-CHO as the sole GSI to study the biological outcomes of blocking Notch signaling [41][42][43] should be interpreted cautiously or reproduced using more specific GSIs. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "The present study demonstrated that the cytotoxicity of Z-LLNle-CHO toward breast cancer cells was not mediated by γ-secretase inhibiton as reported previously, but by proteasome inhibition.This clarification might help its potential development as a chemotherapeutic agent.The results presented also call for careful interpretation of data produced with using Z-LLNle-CHO as the sole γ-secretase inhibitor. ",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "This project has been made possible through a grant from the Alberta Cancer Research Institute (ACRI) and the Alberta Cancer Foundation.Jianxun Han is supported by an ACRI Graduate Studentship and the CIHR Translation Research Training in Cancer program.Michael Hendzel is an Alberta Heritage Foundation for Medical Research Senior Scholar.We would like to thank Dr Gordon Chan for sharing reagents and helpful discussions and Bonnie Andrais for tissue culture.We are grateful to Dr Xuejun Sun and the Cell Imaging Facility at Cross Cancer Institute for technical support. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests. JH participated in the conception of the study and its design, performed most of the experiments, and wrote the first draft of the manuscript.IM performed cell viability/proliferation assays.MJH and JAT participated in the conception of the study and its design and drafted the final manuscript.All authors read and approved the final manuscript. ",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests. ",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "JH participated in the conception of the study and its design, performed most of the experiments, and wrote the first draft of the manuscript.IM performed cell viability/proliferation assays.MJH and JAT participated in the conception of the study and its design and drafted the final manuscript.All authors read and approved the final manuscript. ",
"section_name": "Authors' contributions",
"section_num": null
}
] |
10.1186/s13256-021-02780-y
|
Posterior reversible encephalopathy syndrome in a patient with Richter's syndrome on combination DA-R-EPOCH chemotherapy regimen: a case report
|
<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>Posterior reversible encephalopathy syndrome (PRES) is a clinical-radiologic entity characterized by headaches, altered mental status, seizures, visual loss, and a characteristic imaging pattern in brain magnetic resonance images. The exact etiology and pathogenesis of this condition are not yet fully elucidated.</jats:p> </jats:sec><jats:sec> <jats:title>Case presentation</jats:title> <jats:p>A 72-year-old White man presented with 2 weeks of low-grade fever and chills, night sweats, fatigue, dysphagia, and new-onset rapidly increasing cervical lymphadenopathy. He had a history of chronic lymphocytic leukemia with transformation to diffuse large B-cell lymphoma for which he was started on dose-adjusted rituximab, etoposide, prednisone vincristine, cyclophosphamide, and doxorubicin (DA-R-EPOCH). Shortly after treatment initiation, the patient developed severe airway obstruction due to cervical lymphadenopathy that required emergency intubation. A few days later, the cervical lymphadenopathy and the status of the airway improved, and sedation was consequently weaned off to plan for extubation. However, the patient did not recover consciousness and developed generalized refractory seizures. Brain magnetic resonance imaging revealed edema in the cortical gray and subcortical white matter of the bilateral occipital and inferior temporal lobes, consistent with PRES.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>Posterior reversible encephalopathy syndrome refers to a neurological disorder and imaging entity characterized by subcortical vasogenic edema in patients who develop acute neurological signs and symptoms of a usually reversible nature in different settings, including chemotherapy. Despite its name, PRES is not always fully reversible, and permanent sequelae can persist in some patients. Clinicians should be aware of the possible association between chemotherapy and PRES to ensure early recognition and timely treatment.</jats:p> </jats:sec>
|
[
{
"section_content": "The conversion to aggressive lymphoma in patients with chronic lymphocytic leukemia (CLL), or Richter syndrome, is a concerning complication that carries a very poor prognosis, with a usual median survival of between 8 and 21 months [1].The combination chemotherapy regimen of etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and the relatively recent addition of rituximab, also known as R-EPOCH (or DA-R-EPOCH for dose-adjusted regimen), has improved the management and outcome of these hematological malignancies [2].Despite some existing data, at the present time, no single chemotherapeutic agent has been established to have a consistent association with the development of posterior reversible encephalopathy syndrome (PRES), although among the chemotherapy drugs, cisplatin, cytarabine, adriamycin, cyclophosphamide, and the vinca alkaloids have been occasionally implicated with this syndrome [11].Some published data has also linked the DA-EPOCH combination chemotherapy regimen with PRES.Despite its rarity, this condition should be readily recognized and appropriately managed. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "A 72-year-old White man with an 8-year history of CLL previously treated with four cycles of fludarabine, cyclophosphamide, rituximab in 2012, and ibrutinib since 2014, presented to the oncology clinic with 2 weeks of low-grade fever and chills, night sweats, fatigue, dysphagia, and new-onset rapidly increasing cervical lymphadenopathy.The interval workup revealed worsening of lymphocytosis (61% at presentation vs. 40.2%at 6 months before the presentation) and thrombocytopenia (42 vs. 92 K/mm 3 ).A positron emission tomography scan showed bulky confluent hypermetabolic adenopathy throughout all bilateral neck, chest, abdomen, and pelvis portions.The patient was subsequently admitted to the hospital with suspected CLL progression and possible transformation to a more aggressive type.Lymph node biopsy revealed that high-grade diffuse large B-cell lymphoma (DLBCL) had transformed from CLL (Richter syndrome; Fig. 1).The patient was started on DA-R-EPOCH chemotherapy.However, the patient's course was complicated by tumor lysis syndrome, acute kidney injury, and neutropenic fever with Pseudomonas aeruginosa bacteremia.Therapy with broad-spectrum antibacterials and antifungals and with granulocyte colony-stimulating factor (G-CSF) was initiated. Despite an improvement in cervical lymphadenopathy, on day 9 of hospitalization, the patient developed severe airway obstruction and immediately underwent emergency intubation for airway protection, following which he was transferred to the intensive care unit (ICU).Magnetic resonance imaging (MRI) of the neck demonstrated significant neck soft-tissue edema, for which he received high doses of hydrocortisone, but with no improvement.On day 10, the patient remained unresponsive after sedation was weaned off, and he subsequently developed two episodes of generalized seizures.Computed tomography of the head showed no acute abnormalities.An electroencephalogram did not demonstrate any signs of focal or generalized seizure activity.Brain MRI revealed edema in the cortical gray and subcortical white matter of the bilateral occipital and inferior temporal lobes, consistent with posterior reversible encephalopathy syndrome (Fig. 2).The patient experienced recurrent seizure episodes refractory to lorazepam, levetiracetam, valproate, but responsive to midazolam and propofol.Of note, additional blood cultures became positive for Klebsiella oxytoca; thus, tigecycline was added to the antibiotic regimen.However, considering the overall grim prognosis, the decision was made, in conjunction with the family, not to escalate care.Unfortunately, the patient's condition deteriorated further, resulting in cardiac arrest.We concluded that the nature of PRES, in this case, was multifactorial and related to the recently initiated combination chemotherapy, with acute kidney injury and sepsis being essential additional risk factors. ",
"section_name": "Case presentation",
"section_num": null
},
{
"section_content": "Posterior reversible encephalopathy syndrome refers to a neurological disorder and imaging entity characterized by subcortical vasogenic edema in patients who develop acute neurological signs and symptoms of a usually reversible nature in different settings [3,4].This disorder Fig. 1 Lymph node biopsy showing Richter syndrome.The lymph node architecture was effaced by a diffuse proliferation of large lymphoid cells with features of immunoblasts.A starry sky pattern and frequent mitoses were also noted.The large cells were intermixed with a numerically smaller population of small, mature-appearing lymphocytes.The large cells were positive for CD20, CD79a, BCL6, and MUM1 and negative for CD10 and CD3 by immunohistochemistry.The Ki-67 proliferation index was approximately 80%, and in situ hybridization for Epstein-Barr virus was negative.According to Hans' algorithm, these findings are consistent with diffuse large B-cell lymphoma, activated B-cell subtype, arising in a background of chronic lymphocytic leukemia/small lymphocytic lymphoma is characterized by acute or subacute onset of symptoms, including headache, impaired visual acuity, visual field deficits, changes in consciousness, confusion, and focal deficits [3].Seizures can occur in about two-thirds of the patients [5].While epidemiological data suggest an increased prevalence in middle-aged female patients, it can affect all age groups, including older adults such as the patient presented in this case.Accurate epidemiological data are controversial, partly due to the challenges of confirming the diagnosis and subsequent under-identification of the syndrome [6,7]. While not a radiological diagnosis by itself, PRES requires imaging support to complement the clinical context.Imaging findings include vasogenic edema, watershed distribution, and a parieto-occipital pattern with areas of T2 signal abnormality on MR images [3,4].However, the involvement of frontal and temporal lobes is also common.MRI imaging can be useful to distinguish between cytotoxic edema (hyperintensity on imaging) and vasogenic edema (iso-or hypointensity on imaging) [12].It is relevant to mention that fluid-attenuated inversion recovery (FLAIR) has a higher sensitivity as an imaging modality for diagnosing PRES [8]. Although the exact mechanism is unknown, it is believed that cytotoxic drugs disrupt the function of the blood-brain barrier and generate vasogenic edema through endothelial dysfunction and failure of cerebral autoregulation [9].As chemotherapy use keeps increasing, there is a higher likelihood that PRES may be diagnosed more frequently in the future.When medical conditions such as acute hypertension, renal dysfunction, or electrolyte imbalances are present, the risk of developing PRES increases, as illustrated in our case [10,11]. Currently, no single chemotherapeutic agent has been consistently associated with PRES.Chemotherapy drugs that have been occasionally implicated include cisplatin, cytarabine, adriamycin, cyclophosphamide, and the vinca alkaloids [11].Floeter et al. reported that the DA-EPOCH regimen caused PRES in three of 44 patients (7%) in a retrospective study at their institution [12].Several risk factors, including pre-existing central nervous system (CNS) insult, changes in the fluid status, electrolyte abnormalities, and hypertension, were considered in their analysis.However, their study included a small number of patients, and no reported cases involved the addition of rituximab to the therapeutic regimen [12].These limiting factors leave undetermined if the addition of rituximab makes a difference in the likelihood of PRES occurrence. PRES management is mostly symptomatic, with blood pressure and seizure management being essential components [4].When secondary to cytotoxic medications, such as chemotherapy, it is still controversial whether tapering off or suddenly discontinuing chemotherapy is beneficial.One study compared three interventions after tacrolimus-induced PRES in pediatric patients: one group continued taking the same dose of tacrolimus, the second group discontinued the treatment altogether, and the third group was switched to another agent.No differences in mortality were identified [13].Further studies are needed to identify if a similar scenario occurs in patients receiving chemotherapy. Despite its name, PRES is not always fully reversible, and long-lasting sequelae can persist in 10-20% of patients [14].The prognosis is usually favorable, and most of the patients fully recover.In our patient, mortality was likely driven by the development of bacteremia, septic shock, and progressive renal failure rather than a neurological etiology. To our knowledge, this report represents the first description of DA R-EPOCH combination chemotherapy associated with PRES.In addition to the chemotherapy, the patient also had progressively worsening acute kidney injury and sepsis as risk factors, which may have increased the likelihood of developing this neurological condition and further deterioration leading to death.More research is required to identify the incidence and the strength of association of PRES with various chemotherapy regimens, especially DA R-EPOCH, and to delineate the populations at risk.Clinicians should be aware of the possible association between chemotherapy and PRES to ensure early recognition and timely treatment. ",
"section_name": "Discussion and conclusion",
"section_num": null
}
] |
[
{
"section_content": "None. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "The data used to support the findings of this study are available from the corresponding author on request, except for the patient's personal health information due to Health Insurance Portability and Accountability regulations. ",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "CLL: Chronic lymphocytic leukemia; CNS: Central nervous system; DA-R-EPOCH: Dose-adjusted rituximab, etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin; DLBCL: Diffuse large B-cell lymphoma; FLAIR: Fluid-attenuated inversion recovery; G-CSF: Granulocyte colony-stimulating factor; ICU: Intensive care unit; MRI: Magnetic resonance imaging; PRES: Posterior reversible encephalopathy syndrome. Authors' contributions GE: conceptualization, visualization, writing of original draft, review, and editing.RM-F: conceptualization, writing of original draft, review, and editing.MA: writing of review and editing.NA-G: writing of review and editing.MAY-B: writing of review and editing.BP: writing of review and editing.DPT-G: writing of review and editing.RI: resources.DF: resources.AG: conceptualization and resources.GR-N: supervision, project administration, writing of review and editing.All authors read and approved the final manuscript. Ethics approval and consent to participate Our institution does not require ethical approval for case reports. Written informed consent was obtained from the patient's next of kin for publication of this case report and any accompanying images.A copy of the written consent is available for review by the Editor-in-Chief of this journal. The authors declare that they have no conflicts of interest. • 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 Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "CLL: Chronic lymphocytic leukemia; CNS: Central nervous system; DA-R-EPOCH: Dose-adjusted rituximab, etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin; DLBCL: Diffuse large B-cell lymphoma; FLAIR: Fluid-attenuated inversion recovery; G-CSF: Granulocyte colony-stimulating factor; ICU: Intensive care unit; MRI: Magnetic resonance imaging; PRES: Posterior reversible encephalopathy syndrome. Authors' contributions GE: conceptualization, visualization, writing of original draft, review, and editing.RM-F: conceptualization, writing of original draft, review, and editing.MA: writing of review and editing.NA-G: writing of review and editing.MAY-B: writing of review and editing.BP: writing of review and editing.DPT-G: writing of review and editing.RI: resources.DF: resources.AG: conceptualization and resources.GR-N: supervision, project administration, writing of review and editing.All authors read and approved the final manuscript. ",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "Ethics approval and consent to participate Our institution does not require ethical approval for case reports. ",
"section_name": "Declarations",
"section_num": null
},
{
"section_content": "Written informed consent was obtained from the patient's next of kin for publication of this case report and any accompanying images.A copy of the written consent is available for review by the Editor-in-Chief of this journal. ",
"section_name": "Consent for publication",
"section_num": null
},
{
"section_content": "The authors declare that they have no conflicts of interest. • 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": "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/s13287-019-1285-y
|
miR-34a promotes bone regeneration in irradiated bone defects by enhancing osteoblastic differentiation of mesenchymal stromal cells in rats
|
Radiation exposure negatively affects the regenerative ability and makes reconstruction of bone defects after tumor section difficult. miR-34a is involved in radiation biology and bone metabolism. The aim of this study was to investigate whether miR-34a could contribute to bone regeneration in irradiated bone defects.The expression of miR-34a was analyzed during the osteoblastic differentiation of irradiated BMSCs and bone formation in irradiated bone defects. miR-34a mimics and miR-34a inhibitor were used to upregulate or suppress the expression of miR-34a in BMSCs irradiated with 2 or 4 Gy X-ray radiation. In vitro osteogenesis and subcutaneous osteogenesis were used to assess the effects of miR-34a on the osteogenic ability of radiation-impaired BMSCs. Collagen-based hydrogel containing agomiR-34a or antagomiR-34a were placed into the 3-mm defects of irradiated rat tibias to test the effect of miR-34a on bone defect healing after irradiation.miR-34a was upregulated in the process of bone formation after irradiation. Transfecting radiation-impaired BMSCs with miR-34a mimics enhanced their osteoblastic differentiation in vitro by targeting NOTCH1. Overexpression of miR-34a enhanced the ectopic bone formation of irradiated BMSCs. In situ delivery of miR-34a promoted bone regeneration in irradiated bone defects.miR-34a promoted the osteoblastic differentiation of BMSCs and enhanced the ectopic bone formation after irradiation. miR-34a promoted bone defect healing in irradiated rat tibias. miR-34a-targeted therapy might be a promising strategy for promoting the reconstruction of bone defects after radiotherapy.
|
[
{
"section_content": "Surgical procedures and radiotherapy are critical components of the multidisciplinary management of bone and soft tissue tumors [1].The resection of tumors can result in extensive bone defects, and reconstruction of the defect is needed [2].However, radiotherapy hampers wound healing during bone reconstruction and restoration [3], which lead to higher rates of flap losses [4] and implant failures [5] in patients with radiotherapy. While destroying potential residual malignant cells, radiotherapy also damages adjacent normal tissues [6].The hypocellularity, hypovascularity, and hypoxia condition after irradiation inhibits bone growth and healing [7].In irradiated bone, fewer osteoblast cells and osteocytes are observed, which impedes osteogenesis and bone mineralization [6].Bone marrow mesenchymal stromal cells (BMSCs) are important source of osteoblasts, but the osteoblastic differentiation of BMSCs is impaired by irradiation [8,9].It is necessary to discover the molecular mechanisms regulating osteogenic differentiation of BMSCs under irradiated conditions.miR-34a is known as a tumor suppressor that represses the invasion, metastasis, and drug resistance of malignant cells [10].miR-34a is also involved in tissue development and non-cancerous diseases [11].It is reported that miR-34a plays a role in regulating osteogenic differentiation of stem cells and bone metabolism [12][13][14].Irradiation increases the expression of miR-34a in malignant cell lines and normal cells or tissues [15].However, the function of miR-34a in bone formation after irradiation is unknown. In this study, we observed that miR-34a was upregulated during the osteoblastic differentiation of irradiated BMSCs and bone formation in irradiated bone defects.Overexpression of miR-34a improved the osteoblastic differentiation of irradiated BMSCs and promoted bone regeneration in bone defects of irradiated rat tibias.miR-34a exerted this effect by repressing NOTCH1.Our findings indicated that miR-34a might be a promising therapeutic target for promoting the reconstruction of the bone defect after malignant tumor resection. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "All animal experiments were approved by the Animal Care Committee of Fourth Military Medical University (Permit Number: kq-022), and all the experimental procedures were conducted following the relevant guidelines and regulations.Sprague-Dawley rats of 2 weeks old or irradiated rats were used for the isolation of BMSCs.Six female nude mice of 6 weeks old were used in the ectopic osteogenesis experiment.Male Sprague-Dawley rats weighing 240-270 g were used for irradiation.Eighteen male Sprague-Dawley rats were used for comparison of miRNA expression and bone regeneration in irradiated and non-irradiated bones.Twelve male Sprague-Dawley rats were used to study the enhancement of miR-34a on bone repair in irradiated bones. ",
"section_name": "Ethics statement",
"section_num": null
},
{
"section_content": "Rat BMSCs were isolated and cultured as described before [16].Briefly, bone marrow was flushed from tibias and femurs after all soft tissues were removed.The bone marrow suspension was fractionated by density gradient centrifugation (Histopaque-1083, Sigma, MO, USA) for 25 min at 400 g, and the mononuclear cells were used.Cells were cultured in α-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS; Sijiqing, Hangzhou, China) and 1% penicillin and streptomycin.Cells of passage 2 were used in experiments.Sprague-Dawley rats of 2 weeks old were used for the isolation of BMSCs for in vitro test and subcutaneous osteogenesis.BMSCs from irradiated rats were isolated 8 weeks after irradiation. For osteogenic differentiation, 1 × 10 5 /well BMSCs were seeded in 12-well plates.Cells were cultured in an osteogenic medium containing 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate, and 10 nM dexamethasone.Alkaline phosphatase (ALP) staining was tested by BCIP/NBT ALP color development kit (Beyotime, Shanghai, China), and intracellular ALP activity was tested by ALP assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocol after induction for 7 days.Extracellular matrix mineralized nodules were tested by 1 wt% Alizarin Red S staining (Leagene Biotechnology, Beijing, China) after induction for 21 days.Alizarin Red S staining was further quantitatively analyzed by being dissolved in 10% cetylpyridinium chloride.The absorbance values were measured at 560 nm.Osteogenesis related mRNA and protein expression were tested after 14 days of osteogenic induction by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. ",
"section_name": "Culture of rat BMSCs and osteoblastic differentiation",
"section_num": null
},
{
"section_content": "When BMSCs reached 95% confluence, cells were irradiated with 0, 2, 4, and 8 Gy of X-ray radiation delivered at a rate of 1.1 Gy/minute (RS-2000 XE Biological Irradiator, Rad Source Technologies, GA, USA).The culture medium was replaced by the osteogenic medium after irradiation. ",
"section_name": "Irradiation of BMSCs",
"section_num": null
},
{
"section_content": "The BMSCs were transfected with miR-34a mimics (50 nM), mimics control (50 nM), miR-34a inhibitor (100 nM), inhibitor control (100 nM), small interfering RNA (siRNA) targeting Notch1 mRNA (50 nM), or negative control (50 nM) (Ruibo, Guangdong, China) using Lipofectamine 2000 after irradiated by 2 or 4 Gy.The medium was replaced by the osteogenic medium 6 h after transfection. ",
"section_name": "Transfection",
"section_num": null
},
{
"section_content": "Total RNA was extracted by TriZol (Invitrogen, CA, USA), and 500 ng total RNA was transcribed into cDNA by a PrimeScript RT reagent kit (TaKaRa, Kyoto, Japan).qRT-PCR was performed with SYBR PremixExTaq™II (TaKaRa) on the CFX96™Real Time RT-PCR System.Relative expression was calculated by the ΔΔCt method, and Gapdh was used for normalization.The primers were synthesized as shown in Table 1.For miRNA quantification, Bulge-loopTM qRT-PCR Primer Sets (one PT primer and a pair of qPCR primers for each set) specific for miR-34a and U6 were designed by Ruibo. ",
"section_name": "RNA extraction and quantitative real-time PCR (qRT-PCR)",
"section_num": null
},
{
"section_content": "Cells were lysed in RIPA buffer containing a protease inhibitor cocktail (Sigma, MO, USA).Protein concentrations were quantified by the BCA protein assay (Beyotime).Forty micrograms of protein of each sample was loaded on 10% SDS-PAGE and transferred to the PVDF membranes after separation.The membranes were blocked with 5% BSA for 2h and incubated with primary antibodies for rat COL-1(Protein tech, 14695-1-AP), RUNX2 (Santa Cruz Biotechnology, sc-10758), ALP (Protein tech, 11187-1-AP), osteocalcin (OCN; Santa Cruz Biotechnology, sc-390877), NOTCH1 (Cell Signaling Technology, #4380), and GAPDH (Abcam, ab8245) overnight at 4 °C.The membranes were incubated for 2 h with secondary antibodies (Cowin Biotech, China).The protein bands were incubated with a chemiluminescence kit (Amersham Biosciences, USA) and visualized by the imaging system (Tanon 5500, China).The exposure time is 20 s for GAPDH and 100-200 s for other protein bands depending on the obtained signal intensity.The gray value of the protein bands was quantified by using Image-Pro Plus 6.0 software and normalized to that of GAPDH before comparison. ",
"section_name": "Western blot analysis",
"section_num": null
},
{
"section_content": "For subcutaneous osteogenesis test, 2 × 10 5 /well BMSCs were seeded in six-well plates and treated with growing medium containing 50 μg/ml vitamin C for 5 days.BMSCs were irradiated with 2 Gy and 4 Gy of X-ray radiation and transfected with miR-34a mimics, mimics control, miR-34a inhibitor, and inhibitor control as described above.The medium was replaced by osteogenic medium 6 h after transfection and further cultured for 3 days.The cultured cells along with their deposited extracellular matrix (ECM) were detached as intact sheets from the dishes (BMSC-sheets).Four layers of BMSC-sheets were composited with three layers of bone substitute (20 mg in total, particles 0.25-1 mm, Geistlich) and packaged as a block mass for subcutaneous transplantation as previously described [17].The implants composed of cell sheets and bone substitute were subcutaneously transplanted into the backs of nude mice (n = 3) .Samples were harvested 8 weeks after implantation for histological assay.The implants were fixed by 4% paraformaldehyde for 48 h and decalcified with 18% EDTA for 4 weeks and prepared for H&E staining. ",
"section_name": "Subcutaneous osteogenesis model",
"section_num": null
},
{
"section_content": "Rats were fixed in a perspex jig with their tibias extended laterally.The tibias of rats were subjected to irradiation, and other parts of the body were protected with lead shielding.A single dose of 15 Gy of X-ray radiation was delivered at a rate of 1.1 Gy/minute (RS-2000 XE Biological Irradiator, Rad Source Technologies). ",
"section_name": "Irradiation of rats",
"section_num": null
},
{
"section_content": "For comparison of miR-34a expression and bone regeneration in irradiated and non-irradiated bones, the right tibia of each rat was irradiated.Bone defect surgeries were conducted 3 weeks after irradiation.A 3-mm tibial defect was generated in both tibias.Rats were sacrificed at 2, 4, and 8 weeks after surgery, and the tibias were harvested.For the test of miR-34a expression, the newly formed bone in the defect area was harvested and ground in liquid nitrogen (n = 3).The total RNA was extracted with Trizol reagent (Invitrogen), and miRNA expression was evaluated by qRT-PCR.Newly formed bone was evaluated by Micro-CT (Y.Cheetah, Y.XLON, Hamburg, Germany) and H&E staining (n = 3). To study the enhancement of miR-34a on bone repair in irradiated bone defects, we subjected both tibias of each rat to irradiation.A 3-mm tibial defect was generated in both tibias.Two hundred fifty picomoles of agomir-34a and antagomir-34a, or equal amounts of their respective negative controls (3 μl), were dispensed into 15 μl Col-Tgel component A and 1.5 μl component B (Bioruo, Beijing, China), and then were implanted within the defects.At 8 weeks post-implantation, bone regeneration in the defect was evaluated using Micro-CT (Y.Cheetah, Y.XLON) and histological techniques as described below. ",
"section_name": "Rat tibial defect model",
"section_num": null
},
{
"section_content": "Tibias were harvested and fixed in 4% paraformaldehyde and scanned by Micro-CT (Y.Cheetah, Y.XLON).Scan settings were rotation of 360°, rotation step of 0.5°, kilovoltage of 80 kV, current of 62 μA, and scanning resolution of Table 1 Primers used for qRT-PCR Gene Forward primer sequence(5′-3′) Reverse primer sequence(5′-3′) Ocn 5′-AGG GCA GTA AGG TGG TGA AT-3′ 5′-GCA TTA ACC AAC ACG GGG TA-3′ Col-1 5′-GCC TCC CAG AAC ATC ACC TA-3′ 5′-GCA GGG ACT TCT TGA GGT TG-3′ Gapdh 5′-GGCACAGTCAAGGCTGAGAATG-3′ 5′-ATGGTGGTGAAGACGCCAGTA-3′ 18 μm.VG StudioMAX (Volume Graphics, Heidelberg, Germany) was used for reconstructing 3D images and data analysis.The original bone defect area (L, 2 mm; φ, 3 mm) was defined as the region of interest (ROI).The percentages of BV/TV were calculated to compare the bone regeneration (n = 5). ",
"section_name": "Test of micro-CT",
"section_num": null
},
{
"section_content": "Tibias were decalcified for 4 weeks in 18% EDTA (pH 7.0) and embedded in paraffin.Sections (5 µm thickness) were cut and stained with H&E according to standard protocols. ",
"section_name": "H&E staining",
"section_num": null
},
{
"section_content": "To assess bone formation and mineralization in bone defect area, different fluorochromes were injected intramuscularly at a sequence of 30 mg/kg Alizarin Red S (Sigma), 20 mg/kg Calcein (Sigma), and 20 mg/kg Tetracycline Hydrochloride (Sigma) at 3, 5, and 7 weeks after the operation.Tibias were embedded in polymethylmethacrylate (PMMA) and cut into 50-μm sections (LEICA SP1600, Wetzlar, Germany).The undecalcified sections were observed using a confocal laser scanning microscope (OLYMPUS FV1000, Tokyo, Japan), and the area of fluorescent labelling was quantified by Image-Pro Plus 6.0 software (n = 3). ",
"section_name": "Sequential fluorescent labelling assay",
"section_num": null
},
{
"section_content": "Results were displayed as mean ± standard deviation at least three biological replicates.Differences between two groups were determined by the Student's t test.Differences among groups were analyzed by one-way ANOVA followed by Tukey's post-test.GraphPad Prism7 software was used, and statistical significance was considered when p < 0.05. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To verify whether miR-34a is involved in osteoblastic differentiation of radiation-impaired BMSCs, we subjected BMSCs to different doses of X-ray radiation and tested the osteogenesis and the expression of miR-34a.The cultured BMSCs showed typical BMSC characterizations (Additional file 1: Figure S1).X-ray radiation inhibited the osteoblastic differentiation of BMSCs.Both Alp (Fig. 1a) and Alizarin Red S (Fig. 1b) staining were decreased after irradiation.Irradiation caused a reduction in mRNA expression of Alp, Col-1, and Ocn (Fig. 1c).The protein levels of ALP and COL-1 were decreased in irradiated cell.RUNX2 protein levels of the 0 Gy group were higher than the 8 Gy group.OCN protein levels of the 0 Gy and 2 Gy group were higher than 4 Gy and 8 Gy group.(Fig. 1d,e).The expression of miR-34a was increased in the 4 Gy and 8 Gy group 24 h post-irradiation (0 day after osteogenic induction) compared to the 0 Gy group.The expression of miR-34a was higher in the 4 Gy and 8 Gy group than 0 Gy group 7 days after osteoblastic differentiation.The expression of miR-34a was higher in all the irradiated groups than 0 Gy group 14 days after osteoblastic differentiation (Fig. 1f). We also tested the osteogenesis and the miR-34a expression in BMSCs isolated from irradiated (IR-BMSC) and non-irradiated (N-BMSC) rat tibias.IR-BMSC showed lower expression of Runx2, Alp, Col-1, and Ocn than N-BMSC 14 days after osteogenic induction (Fig. 1g).The IR-BMSC exhibited higher miR-34a level than N-BMSC after osteoblastic differentiation (Fig. 1h). ",
"section_name": "The expression of miR-34a increased in irradiated BMSCs after osteoblastic differentiation",
"section_num": null
},
{
"section_content": "Since miR-34a level was higher in the irradiated BMSCs than the non-irradiated BMSCs after osteoblastic differentiation in vitro, we further investigated whether miR-34a was involved in bone formation in vivo after irradiation.Irradiation inhibited bone formation in the defect area (Fig. 2a).The primary callus formation was postponed by irradiation, as the BV/TV of cortical and trabecular bone were lower in the irradiated group than the non-irradiated group at 2 weeks (Fig. 2b,c).Irradiation caused a delay in the osseous closure, as the newly formed cortical bone was less in the irradiated group than in the non-irradiated group at 4 weeks (Fig. 2b).Subsequent remolding was also reduced in the irradiated group, as the BV/TV of trabecular bone was higher in the irradiated group than the nonirradiated group at 4 and 8 weeks (Fig. 2c).The expression level of miR-34a in the newly formed bone was higher in the irradiated group than the non-irradiated group at 2, 4, and 8 weeks after bone defect surgery (Fig. 2d). ",
"section_name": "The expression of miR-34a increased in newly formed bone after irradiation",
"section_num": null
},
{
"section_content": "To evaluate the effect of miR-34a on osteoblastic differentiation of irradiated BMSCs, we transfected 2 Gy or 4 Gy irradiated BMSCs with miR-34a mimics, miR-34a inhibitor or their controls.The transfection efficiency was estimated to be 75-90%.Two days after transfection, miR-34a showed a > 150-fold increase in the miR-34a mimics group and > 38% reduction in the miR-34a inhibitor group compared with their negative controls.The expression levels remained an 80-fold increase in the miR-34a mimics group and > 30% reduction in the inhibitor group compared with their negative controls after 14 days of osteogenic induction (Additional file 1: Figure S2). For 2 Gy irradiated BMSCs, Alp staining and alizarin red staining were increased in the miR-34a mimics group and decreased in the miR-34a inhibitor group compared with their control groups (Fig. 3a).The Alp activity was enhanced in the miR-34a mimics group compared with the control group (Fig. 3a).The expression of RUNX2, ALP, COL-1, and OCN at both mRNA and protein levels were enhanced by miR-34a overexpression and reduced by miR-34a suppression (Fig. 3b-d).Similar tendency was found in the 4 Gy irradiated BMSCs (Additional file 1: Figure S3). ",
"section_name": "miR-34a overexpression enhanced the osteoblastic differentiation of irradiated BMSCs in vitro",
"section_num": null
},
{
"section_content": "Notch signaling in bone marrow suppresses the osteoblast differentiation of BMSCs [18].NOTCH1 was previously identified as a direct target of miR-34a in tumor cells [19,20].The relationship between miR-34a and NOTCH1 was previously confirmed in human adipose-derived stem cells during osteoblastic differentiation process [14].To confirm whether miR-34a promotes osteoblastic differentiation of irradiated BMSCs through regulating NOTCH1, we tested the expression of NOTCH1 in irradiated BMSCs after osteogenic induction.The mRNA expression of Notch1 of 2, 4, and 8 Gy irradiated BMSCs was higher than the nonirradiated BMSCs (Fig. 4a).The protein expression of NOTCH1 was higher in the 4 Gy and 8 Gy group than in the 0 Gy group.NOTCH1 protein expression was higher in the 8 Gy group than in the 2 Gy group (Fig. 4c).NOTCH1 protein expression was downregulated in the miR-34a mimics group and upregulated in the miR-34a inhibitor group compared with their negative controls (Fig. 4d).Notch1 mRNA expression did not show this trend (Fig. 4b). To further confirm the interaction between NOTCH1 and the osteoblastic differentiation of BMSCs after irradiation, we transfected irradiated BMSCs with siR-Notch.The transfection efficiency was about 80%, and Notch1 mRNA expression was reduced by siR-Notch1 2 days after transfection (Additional file 1: Figure S4).After osteogenic induction, the NOTCH1 expression remained decreased in siR-Notch1 groups compared with the control groups (Fig. 4e).Downregulation of NOTCH1 enhanced the mRNA and protein expression of RUNX2 and OCN (Fig. 4e,f ).Taken together, these data indicated that miR-34a played a role in regulating osteogenesis under irradiated conditions through NOTCH1. ",
"section_name": "miR-34a regulated the osteoblastic differentiation of irradiated BMSCs by repressing NOTCH1",
"section_num": null
},
{
"section_content": "To investigate whether miR-34a could improve the ectopic bone formation of irradiated BMSCs, BMSCs irradiated with 2 Gy or 4 Gy were transplanted subcutaneously into nude mice for 8 weeks after transfected with miR-34a mimics, miR-34a inhibitor, or their negative controls.H&E staining showed that bone formation was enhanced by miR-34a overexpression (Fig. 5a).The percentage of bone area to total area (BA/TA) was used to quantify and compare the amount of newly formed bone.BA/TA was increased by miR-34a overexpression and decreased by miR-34a suppression (Fig. 5b,c). ",
"section_name": "miR-34a overexpression enhanced the ectopic bone formation of irradiated BMSCs",
"section_num": null
},
{
"section_content": "To determine whether miR-34a could enhance bone formation in irradiated bone defects, we established a 3-mm tibial bone defect model 3 weeks after irradiation.The collagen-based hydrogel containing agomir-34a, antagomir-34a, or equal amounts of their respective negative controls were placed into the defect sites.New bone formation in the defect was assessed by micro-CT 8 weeks after surgery.More regenerated bone was observed in the agomiR-34a group compared with the negative control group (Fig. 6a).BV/TV was higher in the agomiR-34a group than the negative control group (Fig. 6d).The antagomiR-34a group showed less regenerated bone and decreased BV/TV compared with the negative control group (Fig. 6a,d).HE staining (Fig. 6b) confirmed the results of the micro-CT scanning. Sequential fluorescent labelling was used to measure bone mineralization and deposition during bone defects repair (Fig. 6c), and the labelling area was quantified (Fig. 6e).Alizarin Red S and Calcein labelling area in the agomiR-34a group were larger than in the agomiR control group, while Tetracycline Hydrochloride area in the agomiR-34a group was less than in the agomiR control group.These results indicated that bone formation and subsequent remolding was enhanced by agomiR-34a.Calcein labelling and Tetracycline Hydrochloride area in the antagomiR-34a group were less than in the agomiR control group, indicating that bone defect repair in irradiated tibias was delayed by antagomiR-34a. ",
"section_name": "miR-34a overexpression enhanced bone formation in irradiated bone defects",
"section_num": null
},
{
"section_content": "The inhibitory effects of radiotherapy on bone formation hinder the reconstruction of bone defects after the resection of bone and soft tissue tumors.In this study, we found a novel strategy based on miRNA to promote bone formation after irradiation.We identified that miR-34a was upregulated during the process of osteogenesis after irradiation.We showed that overexpression of miR-34a promoted the osteoblastic differentiation of irradiated BMSCs.Based on ectopic osteogenesis and tibial defect model, we certified that miR-34a overexpression improved bone regeneration under irradiated conditions. An increasing number of researches have focused on the osteogenesis and miRNA function, both under the biological and pathological conditions.miR-33-5p is a key factor in the development of osteopenia induced by mechanical unloading, and miR-33-5p-targeted treatment could alleviate the osteopenia [21].miR-26a was negatively correlated with bone loss in osteoporotic mice, and miR-26a could be used to restore the osteogenic capacity MSCs under osteoporotic circumstance [22].However, the role of miRNAs in regulating bone formation after irradiation is poorly understood.We found that miR-34a was expressed at a higher level in irradiated BMSCs compared with non-irradiated BMSCs after osteogenic induction.The expression of miR-34a in the regenerated bone within the defect area was also higher in irradiated group than non-irradiated group. Opinions about the role of miR-34a on osteogenesis are conflicting.Fan reported that overexpression of miR-34a significantly increased the osteogenic differentiation of hASCs [14].Kang showed that miR-34a diminished the inhibitory effect of dexamethasone on osteoblastic differentiation of mMSC [23].However, Chen demonstrated that miR-34a is a negative regulator of osteoblast differentiation in hMSC [24].Dang also showed that inhibition of miR-34a suppressed murine arthritis and bone loss [25].The inconsistent results might be due to the different characteristics of cells and the distinct regulation of osteogenesis under biological or pathological conditions.We found that miR-34a improved the osteogenic differentiation of irradiated BMSCs in vitro.BMSCs that transfected with miR-34a showed more ectopic bone formation when transplanted into nude mice.miR-34a overexpression also enhanced bone regeneration in irradiated tibial defects.These data suggested that miR-34a could promote osteogenesis under irradiated conditions.Previous studies had verified that NOTCH1 was a direct target for miR-34a by the dual-luciferase reporter assay [20,26].The interaction of miR-34a and NOTCH1 plays a vital role in cell proliferation and apoptosis and is associated with development and disease in some tissues [27].Notch signaling is also important in bone development and regeneration [28].miR-34a plays a critical role in bone homeostasis, partly through modulating NOTCH1 [14,29].Our data demonstrated that irradiation enhanced NOTCH1 expression and decreased osteogenesis in BMSCs.Overexpression of miR-34a in irradiated BMSCs led to decreased NOTCH1 protein expression and increased osteogenesis.Furthermore, this study showed that suppression of NOTCH1 enhanced the expression of RUNX2 and OCN in irradiated BMSCs.Our findings suggested that miR-34a improved the osteogenic differentiation of irradiated BMSCs by suppressing NOTCH1.However, we observed that both of the expression of miR-34a and NOTCH1 were increased in irradiated BMSCs compared with nonirradiated BMSCs.As the interaction of miRNAs with their target genes is dynamic and dependent on many factors [30], it is possibly the availability and abundance of miR-34a that made a difference in the interaction between miR-34a and NOTCH1 expression. Exploring the delivery systems for miRNAs in bone regeneration has gained increased interests.Different miRNA-regulated systems such as systemic injection [31], site-specific injection [32], and scaffold-based delivery were reported.Hydrogels [33], electrospun nanofibers [34], and nanohydroxyapatite scaffold [35] were used for localized delivery of miRNAs.In this research, we used a collagen-based hydrogel for in situ delivery of miR-34a.This method upregulated the expression of miR-34a within the original bone defects for 2 weeks as demonstrated by intracellular uptake of Cy3-labeled agomir (Additional file 1: Figure S5) and qRT-PCR (Additional file 1: Figure S6).We found that miR-34a enhanced the osteogenic differentiation of BMSCs.However, miR-34a could also influence the function of other cells involved in bone healing and regeneration.miR-34a could inhibit osteoclast differentiation of osteoclast precursors and reduce bone resorption [12,36].miR-34a has an antiangiogenic effect on mouse microvascular endothelial cells [37].A cell-specific miRNA delivery system is needed in the future research to detect the mechanism of miR-34a on bone regeneration after irradiation in vivo. Reconstruction of the bone defect after malignant tumor resection is difficult as the healing processes are influenced by the tumor and the treatment strategies including surgery, radiotherapy, or chemotherapy [3].The regenerative medicine strategies of using stem cells, growth factors, and biomaterials to treat bone defects after tumor resection remain controversial for their potential tumor-promoting effects.miR-34a is a tumor suppressor and regarded as a promising therapeutic agent against cancer [38].Thus, using miR-34a-targeted therapy to enhance bone regeneration in the bone defect after malignant tumor resection may also suppress tumor recurrence. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "In conclusion, this study demonstrated that miR-34a was upregulated in the process of bone formation after irradiation.Overexpression of miR-34a in radiation-impaired BMSCs could promote the osteogenic ability both in vitro and in vivo.In situ delivery of miR-34a promoted bone regeneration in bone defects of irradiated rat tibias.Our findings identified that miR-34a might be a promising therapeutic target for promoting the reconstruction of the bone defect after malignant tumor resection. ",
"section_name": "Conclusion",
"section_num": null
}
] |
[
{
"section_content": "The authors would like to thank Yan Li and Bei Li for their suggestions on the design of the study. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Funding This work was supported by Natural Science Basic Research Plan in Shaanxi Province of China (Program No. 2017JZ026) and Independent Research Project of State Key Laboratory of Military Stomatology (Program No. 2017ZB04). ",
"section_name": "",
"section_num": ""
},
{
"section_content": "All data generated or analyzed during this study are included in this published article. ",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "Additional file 1: All animal experiments were approved by the Animal Care Committee of Fourth Military Medical University (Permit Number: kq-022), and all the experimental procedures were conducted following the relevant guidelines and regulations.Animals were kept in specific pathogen-free conditions (SPF) at 26 °C with a 12-h light/dark cycle.They were given a standard pellet rodent diet and water.All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. 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 file",
"section_num": null
},
{
"section_content": "Additional file 1: ",
"section_name": "Additional file",
"section_num": null
},
{
"section_content": "All animal experiments were approved by the Animal Care Committee of Fourth Military Medical University (Permit Number: kq-022), and all the experimental procedures were conducted following the relevant guidelines and regulations.Animals were kept in specific pathogen-free conditions (SPF) at 26 °C with a 12-h light/dark cycle.They were given a standard pellet rodent diet and water.All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. ",
"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
}
] |
10.1007/s12325-016-0398-2
|
A Systematic Review and Network Meta-Analysis to Evaluate the Comparative Efficacy of Interventions for Unfit Patients with Chronic Lymphocytic Leukemia
|
Rituximab plus fludarabine and cyclophosphamide (RFC) is the standard of care for fit patients with untreated chronic lymphocytic leukemia (CLL); however, its use is limited in 'unfit' (co-morbid and/or full-dose F-ineligible) patients due to its toxicity profile. We conducted a systematic review and Bayesian network meta-analysis (NMA) to determine the relative efficacy of commercially available interventions for the first-line treatment of unfit CLL patients.For inclusion in the NMA, studies had to be linked via common treatment comparators, report progression-free survival (PFS), and/or overall survival (OS), and meet at least one of the five inclusion criteria: median cumulative illness score >6, median creatinine clearance ≤70 mL/min, existing co-morbidities, median age ≥70 years, and no full-dose F in the comparator arm. A manual review, validated by external experts, of all studies that met at least one of these criteria was also performed to confirm that they evaluated first-line therapeutic options for unfit patients with CLL.In unfit patients, the main NMA (five studies for PFS and four for OS) demonstrated clear preference in terms of PFS for obinutuzumab + chlorambucil (G-Clb) versus rituximab + chlorambucil (R-Clb), ofatumumab + chlorambucil (O-Clb), fludarabine and chlorambucil (median hazard ratios [HRs] 0.43, 0.33, 0.20, and 0.19, respectively), and a trend for better efficacy versus rituximab + bendamustine (R-Benda) and RFC-Lite (median HR 0.81 and 0.88, respectively). OS results were generally consistent with PFS data, (median HR 0.48, 0.53, and 0.81, respectively) for G-Clb versus Clb, O-Clb, and R-Clb 0.35 and 0.81 versus F and R-Benda, respectively); however, the OS findings were associated with higher uncertainty. Treatment ranking reflected improved PFS and OS with G-Clb over other treatment strategies (median rank of one for both endpoints).G-Clb is likely to show superior efficacy to other treatment options selected in our NMA for unfit treatment-naïve patients with CLL.F. Hoffmann-La Roche Ltd.
|
[
{
"section_content": "Rituximab in combination with fludarabine and cyclophosphamide (RFC) is currently the standard of care for medically fit patients with previously untreated chronic lymphocytic leukemia (CLL) [1][2][3].However, many patients with CLL are in their 70's and beyond before they need to start treatment, and are likely to have a greater co-morbidity burden [4].For this often medically unfit population, RFC is unsuitable, with data from several clinical studies suggesting that the regimen is associated with excessive toxicity (e.g., cytopenias and increased infection rates) relative to the remission rates achieved [5,6]. Other therapeutic options are available for unfit patients with CLL and include chlorambucil (Clb) in combination with an anti-CD20 antibody such as rituximab (R-Clb), obinutuzumab (G-Clb) or ofatumumab (O-Clb), rituximab in combination with bendamustine (R-Benda), dose-reduced fludarabine with cyclophosphamide (FC), and a dose-modified RFC regimen (RFC-Lite) [2][3][4][7][8][9][10].Available data from randomized controlled trials (RCTs) suggest an improvement in efficacy with certain regimens in this setting.For example, significant improvements in progression-free survival (PFS) have been reported in unfit patients with CLL with R-Benda compared with R-Clb in the MaBLe study (median PFS 39.6 versus 29.9 months; hazard ratio [HR] 0.523; 95% confidence interval [CI] 0.339-0.806;P = 0.003) [10], and with G-Clb compared with the equivalent rituximab regimen in the CLL11 study (29.2 versus 15.4 months; HR 0.40; 95% CI 0.33-0.50;P\\0.001) [11]; however, with a limited number of head-to-head treatment comparisons available, the optimal treatment for unfit patients with CLL remains unclear. Network meta-analysis (NMA) allows information from direct head-to-head studies to be combined with information from indirect treatment comparisons to enable estimation of the comparative efficacy of therapies and build a hierarchy of available treatments [12,13]. Furthermore, the outputs of NMA-based comparative effectiveness research can be used in a full economic appraisal of competing interventions to assess the cost-effectiveness [14].The usefulness of NMA has been demonstrated across a range of therapeutic interventions and disease areas including CLL [15][16][17][18][19][20].Naı ¨ve comparison of drug treatments based on data from different studies carries with it a risk of making incorrect conclusions; by assuming the constancy of relative treatment effects (odds ratios or HRs) to link studies, NMA minimizes this risk.We, therefore, conducted a systematic review and Bayesian NMA of data from all RCTs comparing at least two interventional treatments in patients presenting with 'first-line' CLL and/or who were not eligible to receive full-dose fludarabine (F) to determine the relative effects of treatments on PFS and overall survival (OS). ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "",
"section_name": "METHODS",
"section_num": null
},
{
"section_content": "We conducted an initial literature search plus two updates of five databases (Embase ",
"section_name": "Data Source and Searches",
"section_num": null
},
{
"section_content": "The systematic literature search and screening process for trials are described in section B of the supplementary material.Two independent reviewers screened the titles and abstracts of all identified studies to produce a list of potentially relevant studies.They then performed a detailed screening of the full-text versions of these studies to identify the final list of studies for consideration in the analysis. When reviewing the full text, the objective was to identify whether the study inclusion criteria allowed for the enrollment of patients with co-morbidities, such as renal impairment.In addition, during the same full-text review, the baseline characteristics of patients included in the studies were reviewed to identify (if any) the level of co-morbidities.Any discrepancies in the decisions of the two reviewers were resolved by a third independent reviewer. Studies selected for inclusion in the NMA were RCTs comparing first-line treatment in patients with CLL.Also, included studies had to be linked via common treatment comparators, to report PFS and/or OS data, and were required to meet at least one of five 'co-morbidity' inclusion criteria in descending sequence priority: a median cumulative illness rating score (CIRS) of [6, median creatinine clearance B70 mL/min, existing co-morbidities (particularly relating to renal impairment), median age C70 years, and/or no full-dose F in the comparator arm.These five criteria served as the basis for the identification of publications in the literature that may have evaluated first-line treatments for co-morbid/unfit patients with CLL.A final manual review of all the studies that met at least one of these five criteria was performed and then validated by external experts in the field of CLL, to confirm whether the identified studies did, in fact, evaluate the first-line therapeutic options for co-morbid/unfit and/or full-dose F-ineligible patients with CLL. ",
"section_name": "Study Selection",
"section_num": null
},
{
"section_content": "For each selected trial, unadjusted HRs for PFS and OS were extracted into a pre-defined extraction grid to ensure that the data were extracted uniformly and were comparable across studies.Data were independently extracted by two analysts, with their results checked and reconciled by a third-party independent analyst.If HR data were not reported, HRs were estimated using a method that was appropriate for the available published PFS/OS statistic.For example, if the median PFS/ OS was reported, an exponential distribution was assumed and the HR was estimated as the ratio of the median PFS/OS time for the two treatment arms [21].In papers where landmark PFS/OS data were reported (e.g., 3-year PFS), proportionality of hazard functions was assumed and the HR was calculated as HR = lnS 1 (t)/lnS 0 (t), where S 0 (t) and S 1 (t) denote survival estimates at time t for the control and treatment arm, respectively.In only two of the trials, HRs were not reported and were estimated. NMA as presented in this manuscript was based on the natural logarithms of the HRs (lnHR) and standard deviations (SDs).Published CIs or log-rank P values (in two cases where HRs values were not reported) were used to estimate SDs for lnHRs [22]. ",
"section_name": "Data Extraction and Statistical Analysis",
"section_num": null
},
{
"section_content": "The NMA was conducted using a hierarchical, contrast-based model where lnHR i of trial i follows a normal distribution centered at the (unknown) treatment effect with an SD equal to SD i .To deal with three-arm trials, we followed the approach described by Dias et al. [23].Fixed effects (FE) and random effects (RE) models were explored.The latter accounted for between-study variation using a heterogeneity parameter s. The NMA was performed for PFS and OS on two different evidence networks: the main analysis was based on a network of five studies for PFS and four studies for OS, which were selected according to the five pre-defined criteria and expert opinion (base-case analysis); an additional analysis was also conducted based on a secondary evidence network that included an additional three studies (i.e., a total of eight studies for PFS and seven studies for OS that met at least one of the five pre-defined criteria [no expert involvement]).Model inference was conducted within a Bayesian framework (see supplementary material, section C for the full BUGS code) [24].The posterior distributions of the model parameters were obtained using Markov Chain Monte Carlo (MCMC) methods implemented in the software JAGS [25].All analyses were performed using the computing environment R. The R package ''R2jags'' was used for MCMC simulations.Using three chains, the first 10,000 simulations, with a thinning rate of 500, were discarded as burn-in.Parameters were then monitored for a further 1 million simulations, with the same value of thinning, resulting in a total of 2000 MCMC samples per chain.Convergence of the chains was confirmed using trace plots, density plots of treatment effects, and the Gelman and Rubin's diagnostic statistic [24]. For the FE model, the heterogeneity parameter s was assumed to be zero, and all other relevant parameters were equipped with flat priors.For the RE model, a half-normal prior of the form s * HN[(s u /1.96) 2 ] was considered [26].This prior has its mode at 0 and is steadily declining in s, with an upper 95% point at s u . We set s u at 0.25, which yields an informative prior (a heterogeneity parameter of s u = 0.25 translates to HRs ranging from 0.61 to 1.63 [26]).We note that uninformative priors on s are not appropriate in this setting as most comparisons are informed by a single study (see ''Results''). ",
"section_name": "Network Meta-Analysis",
"section_num": null
},
{
"section_content": "HRs with corresponding 95% credible intervals (CrIs).The treatments were ranked in each MCMC simulation, and medians and 95% CrIs of the posterior ranks were reported.Further, posterior probabilities of being the best treatment were obtained as the proportion of simulations in which each treatment had the smallest HR. ",
"section_name": "All results were reported as median posterior",
"section_num": null
},
{
"section_content": "This article is based on previously conducted studies, and does not involve any new studies of human or animal subjects performed by any of the authors. ",
"section_name": "Compliance with Ethics Guidelines",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "The initial literature search and two updates yielded 244 citations published between January 1992 and August 2015 (supplementary material, section B). Following screening and examination of the papers, we selected a total of eight RCTs that met at least one of the five pre-defined criteria: CLL11, CLL5, COMPLEMENT 1, Nikitin, MaBLe, Knauf, CAM307, and CALGB9011 [8][9][10][27][28][29][30][31].According to expert feedback, three of the studies, Knauf, CAM307, and CALGB9011, did not match the typical unfit patient scenario and were considered to have included patients who were more fit compared with the other RCTs. Table 1 summarizes the main characteristics of the included studies.The treatments evaluated in the eight studies included four single agents: F (in two treatment arms), Clb (six treatment arms), alemtuzumab (Alm; one treatment arm) and bendamustine (Benda; one treatment arm), and five combination regimens: G-Clb (one treatment arm), R-Clb (three treatment arms), R-Benda (one treatment arm), RFC-Lite (one treatment arm) and O-Clb (one treatment arm).Eight RCTs reported PFS, and six RCTs reported OS (Cam307 and Nikitin did not report OS).Treatment effects in terms of PFS and OS for the eight studies are summarized in Table 2. ",
"section_name": "Systematic Review and Included Studies",
"section_num": null
},
{
"section_content": "Figure 1 summarizes the network geometries for PFS and OS, showing the included studies and direct treatment comparisons.The studies excluded in accordance with expert opinion are highlighted in red.We consider a full network of eight RCTs (eight for PFS, seven for OS) and a reduced network of five RCTs (trials excluded in accordance with expert opinion; five for PFS, four for OS).The analysis performed on the reduced network represents our main analysis; the full network was used for completeness in an additional analysis. ",
"section_name": "Network Meta-Analysis",
"section_num": null
},
{
"section_content": "Summary of the eight randomized controlled studies evaluating first-line therapy in chronic lymphocytic leukemia selected for inclusion in the network meta-analysis (main and additional analysis) E). ",
"section_name": "Table 1",
"section_num": null
},
{
"section_content": "The analysis was repeated on all eight trials identified using the five F-ineligibility criteria (no expert assessment).Inclusion of an additional three studies made minimal difference to the estimated treatment effects and the rank ordering of treatments (Fig. 3; Table 4).These findings for the additional analysis were consistent for both the FE and RE models.Data for the additional analysis using the RE model are summarized in the supplementary material, sections D and E. For OS, of note was the narrower CrI for the comparison of G-Clb versus F (FE model: HR 0.57, CrI 0.34, 0.95) compared with the wide CrI presented earlier in the main analysis.The higher precision in the additional analysis is a consequence of the combined evidence from the CLL5 and CALGB9011 trials.Studies Knauf [29], Cam307 [30], and Calgb_9011 [31] included in analysis Alm alemtuzumab, Benda bendamustine, Clb chlorambucil, CrI credible interval, F fludarabine, G-Clb obinutuzumab ?chlorambucil, O-Clb ofatumumab ?chlorambucil, OS overall survival, PFS progression-free survival, R-Benda rituximab ?bendamustine, R-Clb rituximab ?chlorambucil, RFC rituximab ?fludarabine ?cyclophosphamide chlorambucil (G-Clb) is likely to be superior to many other including Benda, R-Clb, O-Clb, Alm, F, and Clb in unfit patients with CLL.In addition, G-Clb showed trend for greater efficacy over the regimens RFC-Lite and R-Benda in this setting.Our results for OS were generally consistent with the data on PFS, suggesting beneficial outcomes with G-Clb over other regimens, and were driven by the consistent trend in OS favoring G-Clb in the CLL-11 trial [11,27,32].However, the OS data in our study were associated with greater uncertainty, and this was not unexpected given that the follow-up times for OS were relatively short.We also note that to the best of our knowledge, PFS has not yet been validated as a surrogate endpoint for OS in the first-line treatment of unfit patients with CLL within a meta-analytic framework [33]. Our NMA of PFS (additional analysis) also supported the findings of the Knauf study suggesting that Benda is a more potent chemotherapy, leading to 'deeper' remission than the traditional agent Clb in unfit patients [29].Given this finding, it was of interest to note a trend favoring the combination of obinutuzumab with Clb over rituximab combined with Benda, as shown by the HR estimate for PFS (0.81 CrI 0.49, 1.33). Through the use of pre-defined 'co-morbidity' inclusion criteria combined with expert review, we were able to restrict our analysis to unfit patients with CLL who were likely to meet the unfit definition as described in the CLL11 trial [27].However, information on CLL patient 'fitness'/CIRS is not always reported in the literature, making it difficult to evaluate a study population's level of fitness/co-morbidity; moreover, there is no well-defined surrogate marker for fitness status/co-morbidity.To address this, we used five pre-defined co-morbidity criteria (median CIRS [6, median CrCL B70 mL/min, existing co-morbidities, median age C70 years, and no full-dose F in the comparator arm) to approximate the level of patient fitness/co-morbidity indirectly in each study.Because of the potential heterogeneity of the identified papers, a manual review of the full text of the identified papers was conducted, and the final selection of papers was approved by experts.This final selection led to the exclusion of the following studies from the main analysis: Cam307 (study inclusion criteria included adequate renal and liver function and median age was 59-60 years) [30], Calgb_9011 (F dose appeared to be the full dose, 20 or 25 mg/m 2 intravenously days 1-5 every 28 days) [31], and Knauf (a high percentage of patients in both the Benda [70%] and Clb [65%] arms had a WHO performance score of 0 and a median age of approximately 65 years, therefore, it was unlikely that the patients included were not eligible for full-dose F) [29].It is also important to note that the three excluded studies were conducted at a time when Clb (rather than RFC) was the standard of care even in fit patients, since at that time, no treatment had shown an OS benefit over Clb.Also, these studies were not designed to explicitly enroll unfit patients, their median age was substantially lower than the remaining five studies, and chemotherapy alone is not currently considered a relevant treatment option (even in unfit CLL). An important advantage of NMA over naı ¨ve inter-trial comparisons is that the calculations are based on relative treatment effects (in terms of HRs) rather than absolute effects.Thereby, NMA circumvents the potential incomparability of two studies due to differences in the distributions of measured and/or unmeasured prognostic factors.For example, a naı ¨ve comparison of the R-Benda arm in MaBLe (median PFS 39.6 months) [10] with the G-Clb arm in CLL11 (median PFS 29.2 months) [11] would have led to the of better performance of R-Benda versus G-Clb.This naı ¨ve comparison compares efficacy on an 'absolute' scale and ignores the fact that the common comparator R-Clb in these studies showed substantial discrepancy in terms of median PFS (29.9 months in MaBLe and 15.4 months in the CLL11 study, respectively).The prognostic differences that lead to this study bias between MaBLe and CLL11 may be manifold with differences in methodology of data generation and data read-out as the main drivers.We note that our NMA does not account for potential effect modifiers, which are defined as patient or study characteristics that influence the two treatment arms to a different extent and, therefore, alter the relative treatment effect.A speculative effect modifier could be the difference in the cumulative dose of Clb in MaBLe and CLL11.However, we do not believe that this solely explains the discrepancies seen in median PFS between the two R-Clb arms from MaBLe and CLL11, since there is evidence which points against 'Clb dosing' as an effect modifier of PFS [27,34,35].Admittedly, there is still some debate around the role of Clb dosing on PFS. The results of our NMA should ideally be validated in independent studies, and our findings should be interpreted taking into account a number of limitations.First, a limited number of studies were eligible for inclusion in the analysis.Many of the comparisons were informed by a single trial (e.g., CLL11 was the only trial to compare G-Clb and R-Clb).This limited the statistical assessment of heterogeneity and inconsistency and also precluded the performance of more sophisticated analyses, for example, meta-regression to adjust for potential effect modifiers.Our selection strategy (five criteria and expert opinion) also aimed at increasing the homogeneity of the trials in the network. Nonetheless, the levels of unfitness still varied among the selected trials, which could ultimately have influenced our NMA.Second, the follow-up times for OS (and to a lesser extent PFS) were relatively short; longer follow-up would likely have impacted on the effect size, especially the CrIs.Third, in two trials, assumptions were required to calculate lnHR and SD, and these estimates may have differed from the estimate of HR using whole Kaplan-Meier curves. Our results are comparable with the findings reported by Ladyzynski et al. [16] The results of a second NMA evaluating US Food and Drug Administration/European treatment options for previously untreated patients with CLL ranked RFC highest in terms of efficacy, while all single agents (with the exception of Alm) occupied worst ranks [19].Unlike our NMA, the authors imposed no restrictions in terms of physical fitness or age of the study participants, and full-dose F studies were also included.Furthermore, the analysis did not include important recent trials, for example, CLL11 (G-Clb), MaBLe (R-Benda), and Complement 1 (O-Clb).In an earlier study, Cheng et al. [36] also used similar NMA methodology to analyze treatments that had not been directly compared in terms of PFS in previously untreated patients with CLL.The findings suggested that RFC achieved relatively longer PFS compared with FC, F, Alm, and Clb (76 months versus 23-60 months); however, the data were limited to younger patients (59-65 years) with good performance status and early-stage disease [36].In a multiple-treatment meta-analysis using direct and indirect data based on all available head-to-head RCTs, Terasawa et al. [37] ",
"section_name": "Additional Analysis",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
}
] |
10.1186/s12931-020-01608-5
|
Mutation profile of non-small cell lung cancer revealed by next generation sequencing
|
<jats:title>Abstract</jats:title> <jats:p><jats:bold>Background<jats:italic>:</jats:italic> </jats:bold>Precision therapy for lung cancer requires comprehensive genomic analyses. Specific effects of targeted therapies have been reported in Asia populations, including Taiwanese, but genomic studies have rarely been performed in these populations. <jats:bold>Method: </jats:bold>We enrolled 72 patients with non-small cell lung cancer, of whom 61 had adenocarcinoma, 10 had squamous cell carcinoma, and 1 had combined adenocarcinoma and squamous cell carcinoma. Whole-exome or targeted gene sequencing was performed. To identify trunk mutations, we performed whole-exome sequencing in two tumor regions in four patients. <jats:bold>Results:</jats:bold> Nineteen known driver mutations in <jats:italic>EGFR</jats:italic>, <jats:italic>PIK3CA</jats:italic>, <jats:italic>KRAS</jats:italic>, <jats:italic>CTNNB1</jats:italic>, and <jats:italic>MET</jats:italic> were identified in 34 of the 72 tumors evaluated (47.22%). A comparison with the Cancer Genome Atlas dataset showed that <jats:italic>EGFR</jats:italic> was mutated at a much higher frequency in our cohort than in Caucasians, whereas <jats:italic>KRAS</jats:italic> and<jats:italic> TP53</jats:italic> mutations were found in only 5.56% and 25% of our Taiwanese patients, respectively. We also identified new mutations in <jats:italic>ARID1A</jats:italic>, <jats:italic>ARID2</jats:italic>, <jats:italic>CDK12</jats:italic>, <jats:italic>CHEK2</jats:italic>, <jats:italic>GNAS</jats:italic>, <jats:italic>H3F3A</jats:italic>, <jats:italic>KDM6A</jats:italic>, <jats:italic>KMT2C</jats:italic>, <jats:italic>NOTCH1</jats:italic>, <jats:italic>RB1</jats:italic>, <jats:italic>RBM10</jats:italic>, <jats:italic>RUNX1</jats:italic>, <jats:italic>SETD2</jats:italic>, <jats:italic>SF3B1</jats:italic>, <jats:italic>SMARCA4</jats:italic>, <jats:italic>THRAP3</jats:italic>, <jats:italic>TP53</jats:italic>,<jats:italic> </jats:italic>and <jats:italic>ZMYM2</jats:italic>. Moreover, all ClinVar pathogenic variants were trunk mutations present in two regions of a tumor. RNA sequencing revealed that the trunk or branch genes were expressed at similar levels among different tumor regions.<jats:bold>Conclusions: </jats:bold>We identified novel variants potentially associated with lung cancer tumorigenesis. The specific mutation pattern in Taiwanese patients with non-small cell lung cancer may influence targeted therapies.</jats:p>
|
[
{
"section_content": "Lung cancer is the leading cause of cancer-associated mortality worldwide.An estimated 2.09 million new lung cancer cases and 1.76 million lung cancer-associated mortalities were reported in the GLOBOCAN 2018 database [1].Lung cancer can be divided into two broad categories according to histology: small-cell lung cancer and non-small-cell lung cancer (NSCLC).The latter comprises more than 80-85% of all lung cancers.NSCLC is subdivided into adenocarcinoma (ADC; 60%), squamous cell carcinoma (SqCC; 30-35%), large cell carcinoma, and other rare tumors, including adenosquamous carcinoma [2].Risk factors include tobacco consumption, genetic susceptibility, poor diet, air pollution and occupational exposures, such as asbestos, metals and mixed occupation exposures, silica, polycyclic aromatic hydrocarbons, and diesel exhaust fumes [3].Tobacco smoking is the main risk factor for lung cancer; however, 10-15% of those diagnosed with lung cancer are never-smokers [4].Epidemiologic studies reveal that the proportion of lung cancer in never-smokers is higher in East Asia [5].Neversmoker East Asian females are diagnosed more often with ADC, and these patients exhibit higher treatment Open Access *Correspondence: [email protected] 1 Epigenome Research Center, China Medical University Hospital, 2 Yuh-Der Road, Taichung 404, Taiwan Full list of author information is available at the end of the article response rates to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors [6].Chest radiography, sputum cytology, and low-dose computed tomography (CT) have been used for lung cancer screening.Despite advances in genomic research and targeted therapies, leading to improvements in therapeutic strategies and the clinical outcomes of lung cancer patients [7], the overall 5-year survival rate of lung cancer remains very low (16.8%)[8].The prognosis of lung cancer remains poor because most patients are often diagnosed at an advanced stage. Rapid advancements in next-generation sequencing technology and a better understanding of cancer biology have provided unprecedented opportunities to characterize the genome of human tumors including lung cancer.The Cancer Genome Atlas (TCGA) lung cancer working group has profiled and analyzed 230 ADC and 178 SqCC specimens to identify molecular aberrations at the DNA, RNA, protein, and epigenetic levels [9,10].In ADC, the most common mutations are TP53, KRAS, EGFR, NF1, BRAF, MET, and RIT.Pathway alterations in ADC are involved in RTK/RAS/RAF, mTOR, JAK-STAT, DNA repair, cell regulation, and epigenetic deregulation.Mutations in TP53, CDKN2A, PIK3CA, NFE2L2, KEAP1, CUL3, PTEN, NF1, NOTCH1, 2 and 3, DDR2, and EGFR genes are frequently observed in SqCC.Pathway alterations in SqCC involve squamous differentiation, the oxidative stress response, PIK3CA, DNA repair, cell cycle regulation, and epigenetic deregulation [11].ADC and SqCC show genetic alterations or gene expression differences [12,13].In ADC, the most common therapeutic targets are EGFR and BRAF mutations and ALK and ROS1 rearrangements.Molecular genotyping is now routine in ADC.In SqCC, targeted agents are largely ineffective, and many targeted therapies are currently undergoing clinical trials [14]. To develop a more comprehensive genomic picture of NSCLC, we performed whole-exome sequencing (WES) or targeted gene sequencing (TGS) in 72 Taiwanese patients with NSCLC.In addition, we compared the results with the TCGA NSCLC dataset, which involves mainly Western populations.We also investigated the associations between genetic alterations and clinicopathological features. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "A total of 61 fresh-frozen and 11 formalin-fixed paraffin-embedded (FFPE) specimens were obtained from 72 Taiwanese patients with lung cancer who underwent surgical resection from May 2007 to April 2019 at the China Medical University Hospital.The 72 lung tumors comprised 61 ADCs, 10 SqCCs, and 1 combined ADC and SqCC.DNA from frozen tissues and FFPE specimens was extracted using the QIAamp ® DNA Micro Kit (Qiagen, Heidelberg, Germany) and QIAamp ® DNA FFPE Tissue Kit (Qiagen) according to the manufacturer's instructions.The DNA concentration was quantified using the NanoDrop1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA) and a Qubit Fluorometer (Invitrogen, Carlsbad, CA, USA). ",
"section_name": "Subjects and DNA extraction",
"section_num": null
},
{
"section_content": "TGS was performed using the Qiagen platform with a panel that included either 275 (cat.no.DHS-3501Z) or 72 genes (cat.no.DHS-005Z).DNA libraries were prepared using components from the QIAseq Targeted DNA Panel Kit (Qiagen) and QIAseq Targeted DNA Panel Human Lung Cancer Panel (Qiagen).Briefly, 80 ng DNA was enzymatically fragmented and end-repaired in a 25-μl reaction volume containing 2.5 μl 10 × fragmentation buffer and 5 μl fragmentation enzyme mix.The reaction was carried out at 4 °C for 1 min, 32 °C for 24 min, and 65 °C for 30 min.Next, 10 μl 5 × ligation buffer, 5 μl DNA ligase, and 2.8 μl 25 μM barcoded adapters were added along with enough water to reach a reaction volume of 50 μl.Reaction tubes were then incubated at 20 °C for 15 min.To ensure complete removal of free barcoded adapters, each reaction was purified using 1.4 × (or 1.0 ×) QIAseq beads for two rounds.The purified DNA was then mixed in a 20-μl reaction volume with 10 nM each target primer, 400 nM IL-Forward primer, 1 × TEPCR buffer, and 0.8 μl HotStarTaq DNA polymerase.The PCR protocol was as follows: 95 °C for 13 min; 98 °C for 2 min; six cycles of 98 °C for 15 s and 65 °C for 15 min; and 72 °C for 5 min.Each reaction was cleaned once using 1.4 × (or 1.0 ×) QIAseq beads to remove unused primers.Enriched DNA was combined with 400 nM IL-Index primers, 1 × UPCR buffer, and 1 μl HotStarTaq DNA polymerase in a volume of 20 μl.The universal PCR conditions were as follows: 95 °C for 13 min; 98 °C for 2 min; 20 cycles of 98 °C for 15 s and 60 °C for 2 min; and 72 °C for 5 min.The DNA library was purified once using 1.4 × (or 1.0 ×) QIAseq beads and quantified using Qubit Fluorometric Quantitation (Thermon Fisher Scientific-US, Waltham, MA, USA).Libraries were sequenced on Illumina NextSeq (paired-end, 2 × 150 bp) according to the manufacturer's instructions (Illumina, San Diego, CA, USA).TGS analysis was described in detail in our previous work [15]. ",
"section_name": "TGS and data analysis",
"section_num": null
},
{
"section_content": "A total of 50 ng DNA (based on Qubit quantification) was tagmented by a transposome, followed by clean-up and amplification of the tagmented DNA.A 200-400 bp band was selected, and exome capture was performed using the Nextera Exome Library Preparation Kit (Illumina).The DNA library was quantified using the Qubit 3.0 Fluorometer (Invitrogen) and Agilent 4200 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).Samples were subjected to paired-end sequencing using the Illumina NovaSeq 6000 platform with a 150-bp read length.WES analysis has been described in detail in our previous work [16]. ",
"section_name": "WES and data analysis",
"section_num": null
},
{
"section_content": "Total RNA was extracted from clinical tissue samples using the NucleoSpin ® RNA Kit (MACHEREY-NAGEL GmgH, Düren, Germany) following the manufacturer's instructions.The quality, quantity, and integrity of the total RNA were evaluated using the NanoDrop1000 spectrophotometer and Bioanalyzer 2100 (Agilent Technologies).Samples with an RNA integrity number > 6.0 were used for RNA-seq.An mRNA-focused, barcoded library was generated using the TruSeq strand mRNA Library Preparation Kit (Illumina).The libraries were sequenced on the Illumina Nova Seq 6000 instrument (Illumina), using 2 × 151-bp paired-end sequencing flow cells following the manufacturer's instructions. ",
"section_name": "RNA sequencing (RNA-seq)",
"section_num": null
},
{
"section_content": "Illumina bcl2fastq Conversion Software (v2.20.0.422) was utilized to convert raw sequencing data to fastq format (Illumina).Trimmomatic PE (v0.39) was applied to control the read quality and remove sequencing adapters [17].Reads were discarded if their average quality was < 20 (AVGQUAL:20) and their read length < 105 bp (MINLEN:105).Next, paired quality-controlled reads were aligned to the human genome (GRCh38), and gene expression was quantified using transcripts per million normalization via the HISAT2 (2.1.0)[18] and StringTie (1.3.5)[19] pipelines.To evaluate the similarities between different regions from the same tissue, we applied Spearman's rank correlation of the transcripts per million values of 299 gene signatures [20].A heatmap of the gene expression values was plotted using Morpheus (https :// softw are.broad insti tute.org/morpheus), and the correlation coefficients were visualized using Seaborn, a Python data visualization library (https ://githu b.com/mwask om/ seabo rn). ",
"section_name": "RNA-seq data analysis",
"section_num": null
},
{
"section_content": "All statistical analyses were performed using SPSS software ver.22.0.Chi-squared or Fisher's exact tests were used to compare two categorical variables.Survival analysis was performed using Kaplan-Meier survival plot and log-rank test.A p-value less than 0.05 was considered statistically significant. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "The patients in this study comprised 39 males and 33 females, with a mean age of 62.35 years, of whom 61 had ADC, 10 had SqCC, and 1 had combined ADC and SqCC.Stage I disease was identified in 38 patients, stage II in 12 patients, stage III in 10 patients, and stage IV in 6 patients (Table 1). ",
"section_name": "Patient characteristics",
"section_num": null
},
{
"section_content": "Among the 72 samples, 32 contained driver mutations in well-known cancer genes in NSCLC, such as EGFR (n = 26; E709G, T790M, L858R and non-frameshift deletions of exon 19), and PIK3CA (n = 4; E542K and G1049R).Besides EGFR and PIK3CA, other known mutations were detected in KRAS (n = 4; G12V, G12A, G12D, and Q61H), which have all been reported as driver mutations in lung cancer.In addition, four samples carried known activating mutations in the well-known oncogenes CTNNB1 (n = 3; S33F, S37C and S37F) and MET (n = 1; R1004X and c.3028 + 1G > T).Overall, 34 specimens harbored driver mutations in five cancer genes (EGFR, PIK3CA, KRAS, CTNNB1, and MET), which are canonical driver mutations (Additional file 1: Table S1).These mutations were mutually exclusive, except for four cases of double mutations (n = 2; EGFR and CTNNB1 and n = 2; EFGR and PIK3CA).TP53 was the most frequently S1).Among the 16 TP53 variants, two were novel (S95fs and F212fs). ",
"section_name": "Genomic alterations",
"section_num": null
},
{
"section_content": "To compare the frequency of driver mutations of NSCLC between Taiwanese and Caucasian patients, we obtained all available lung cancer cases (560 ADC and 489 SqCC) from the TCGA dataset.Notable differences from the TCGA data included the frequencues of mutations in EGFR (36.11% vs. 9.82%, p < 0.0001), KRAS (5.56% vs. 15.92%,p = 0.0165), and TP53 (25.00% vs. 69.69%,p < 0.0001).A full comparison of the frequencies of selected gene alterations between the two cohorts is depicted in Fig. 1 and Additional file 1: Table S2. ",
"section_name": "Comparisons between Taiwanese and Caucasian patients with NSCLC",
"section_num": null
},
{
"section_content": "Based on the latest NSCLC guidelines published by the National Comprehensive Cancer Network, clinically relevant genomic alterations were identified in 34 (47.22%)patients (Table 2).As shown in Table 2, the clinically relevant alterations included in EGFR (26, 36.11%),ERBB2 (2, 2.78%), KRAS (4, 5.56%), MET (1, 1.38%), and NTRK1 (1, 1.38%). Among the 26 patients with EGFR mutation patients, only 8 had an additional TP53 mutation, of whom 1 died of a cause unrelated to NSCLC.Among the 7 remaining patients, 3 had good survival outcomes, and 4 did not.We compared the genetic differences between the patients with a good and those with a poor survival outcome.In addition to the EGFR and TP53 mutations, one patient with poor survival harbored a MYC nonframeshift deletion (p.48_48del, rs776629119), and one patient with good survival had an AR non-frameshift insertion (p.L57delinsLQQQ, rs4045402) and a FBXW7 non-frameshift deletion (p.117_117del, rs781154022), and another patient with good survival had a CTNNB1 (p.S37F, rs121913403) mutation. ",
"section_name": "Clinically relevant genomic alterations",
"section_num": null
},
{
"section_content": "Correlations between the genotypes and clinicopathological characteristics are listed in Table 3.The EGFR mutation rate was significantly higher in patients with ADC than in those with SqCC (41.0% vs. 10.0%,p = 0.059).Moreover, the EGFR mutation rate was significantly higher in patients without smoking than in those with smoking (44.7% vs. 20.0%,p = 0.038).No association was found between the EGFR mutation status and sex, age, or tumor stage of the patients.In contrast, the PIK3CA and TP53 mutation rates were significantly higher in patients with SqCC than in those with ADC (30.0% vs. 1.6%, p = 0.008 and 50.0% vs. 21.3%, p = 0.053).Furthermore, the TP53 mutation rate was significantly higher in patients with smoking than in those without smoking (40.0% vs. 17.0%,p = 0.032).No association was found between KRAS or CTNNB1 mutations and any clinicopathological characteristic. We used Kaplan-Meier curve analysis to assess overall survival.In our cohort, PIK3CA mutation was a prognostic of worse overall survival (Fig. 2).There was, however, no significant difference in mortality between EGFR, KRAS, TP53, and CTNNB1 mutations (Additional file 2: Figure S1). ",
"section_name": "Correlations between driver mutations and clinicopathological characteristics",
"section_num": null
},
{
"section_content": "To exame intratumor heterogeneity, we applied multiregion WES in 8 tumor regions from 4 resected tissues (Fig. 3a).In order to determine whether driver genes carry trunk or branch mutations, we identified potential driver mutations among the 299 known cancer driver genes [20].All variants classified as pathogenic in the ClinVar database are trunk mutations present in two tumor regions (Additional file 1: Table S3).Among the 61 predicted pathogenic variants identified from four patients (15,11,16, and 19, respectively), 37 were classified as trunk mutations (11,4,7, and 15, respectively) (Fig. 3b, Additional file 1: Table S4). We further analyzed the variant allele frequencies of the trunk and branch mutations.Generally, the variant allele frequencies in four paired samples suggested that trunk mutations (median: 0.23-0.34%)occurred much more frequently than branch mutations (median: 0.12-0.15%)(Fig. 3c). We also analyzed the expression of the driver genes that carried trunk or branch mutations.Gene expression profiles revealed no differences in driver genes harboring trunk or branch mutations between the two different tumor regions of the four paired samples (Fig. 3d). ",
"section_name": "Identification of trunk or branch driver mutations",
"section_num": null
},
{
"section_content": "We determined the intratumoral expression of 299 driver genes, which were derived from 33 cancer types in the PanCancer dataset [20].We used Spearman's rank correlation to calculate the gene expression correlations between two regions from four tumors each.Two regions from a tumor showed the highest correlation coefficient (Fig. 4). The numbers of differentially expressed genes with a fold change in expression > 4 in the four paired samples were 4 (FGFR2, PRKAR1A, MYC, and MYD88), 1 (MYD88), 4 (KMT2C, GNA11, ALB, and B2M), and 2 (KLF5 and CDKN2A), respectively (Additional file 1: Table S5).Most of the genes showed consistent expression (fold change ≤ 4).There were few differences between the different regions within the same tumor, and we suggest that any differences were due to branch mutations.Thus, the 299-driver gene signature may correctly predict cancer etiology if assessed from a single tumor region. ",
"section_name": "Intratumoral heterogeneity of 299 driver genes",
"section_num": null
},
{
"section_content": "In the present study, we successfully performed comprehensive genomic profiling in tumor specimens from 72 Taiwanese NSCLC patients using WES or TGS.We found that EGFR mutations were more common in patients with ADC, irrespective of sex, age, or tumor stage.PIK3CA and TP53 mutation rates were also higher in patients with SqCC.A comparison of driver gene mutations in our NSCLC patients with the TCGA dataset showed that EGFR was mutated at a much higher frequency in our Taiwanese cohort compared with Caucasians.In contrast, KRAS, BRAF, and TP53, the most common mutated genes in Caucasians, were found in only 5.56, 0, and 25%, respectively, of the Taiwanese NSCLC patients in our study, which is consistent with other studies [12,21].These differences are most likely due to racial and environmental differences. In addition, we also identified 83 novel variants in 72 genes.Eighteen of these have been reported as cancer driver genes (ARID1A, ARID2, CDK12, CHEK2, GNAS, H3F3A, KDM6A, KMT2C, NOTCH1, RB1, RBM10, RYNX1, SETD2, SF3B1, SMARCA4, THRAP3, TP53 and ZMYM2) (Additional file 1: Table S6) [20].Eight driver genes were associated with LUAD and LUSC. ARID1A encodes a member of the SWI/SNF family of proteins.Mutation of ARID1A has been documented in a number of cancers [22], and approximately 8% of lung ADCs contain mutations in ARID1A [23].In this study, we identified a novel ARID1A mutation (p.G933fs) in a patient with SqCC. KDM6A is located on chromosome Xp11 and encodes a tetratricopeptide repeat protein.The protein contains a Jumonji C domain and catalyzes the demethylation of tri/dimethylated histone H3.KDM6A is a tumor suppressor gene in different cancers, including lung SqCC [20].In this study, we identified a novel KDM6A mutation (p.S314X) in a patient with SqCC. NOTCH1, a member of the NOTCH protein family, contains an extracellular domain consisting of multiple epidermal growth factor-like repeats (36) and an intracellular domain consisting of multiple domain types.NOTCH1 is reported to have a bimodal role as a tumor suppressor and an oncogene in several cancers [24,25].In this study, we identified a novel NOTCH1 mutation (p.E2534K) in a patient with SqCC. RB1 encodes a negative regulator of the cell cycle protein and was the first tumor suppressor gene identified.Its tumor suppressive function is due to inhibition of the transcription factor E2F1. Any defect in the RB1 gene causes cells to transition from the G1 to S-phase of the cell cycle [26].RB1 is inactivated in a wide range of cancers, including lung ADC and SqCC.In this study, we identified a novel RB1 mutation (p.I848fs) in a patient with ADC.The I848fs mutation occurs in the C domain, which mediates the interaction with E4F1 [27]. RBM10, a tumor suppressor gene [28,29], encodes a nuclear protein that contains an RNA-recognition motif.RBM10 regulates pre-mRNA splicing in the alternative splicing pathway [30].In this study, we identified a novel RBM10 mutation (p.A410fs) in a patient with ADC. SETD2 encodes a protein that interacts with huntingtin.The protein is a histone methyltransferase responsible for the tri-methylation of lysine 36 on histone H3 (H3K36me3), using H3K36me2 as a substrate [31].SETD2 is a tumor suppressor gene expressed in different cancer types [32].In this study, we identified a novel SETD2 mutation (p.L1525P), present in the AWS domain, in a patient with SqCC. SMARCA4, also known as BRG1, encodes a member of the SWI/SNF family, which possesses helicase and ATPase activities.Human SWI/SNF enzyme subunits are mutated in approximately 20% of cancers [33].SMARCA4 is possibly tumor supressive in lung ADC.In contrast, SMARCA4 may also be an oncogene in liver hepatocellular carcinoma, lower-grade glioma, and pan-cancer [20].In this study, we identified a novel SMARCA4 mutation (p.Q570fs) in a patient with ADC. Recently, Skoulidis et al. reported that co-occurring genomic alterations affect the response of NSCLC to anticancer therapies [34].The mean overall survival was 82.30 ± 9.80 months for the EGFR mutant TP53 wildtype cohort, 86.88 ± 15.41 months for the EGFR/ TP53-mutant cohort; p = 0.839 (Additional file 2: Figure S2).Among them, 11 patients used EGFR-TKI therapy.We also assessed whether there are differences in survival after EGFR-TKI therapy.The mean overall survival was 58.29 ± 10.26 months for the EGFR mutant TP53 wildtype cohort (n = 8), 114.50 ± 2.50 months for the EGFR/TP53-mutant cohort (n = 3); p = 0.216 (Additional file 2: Figure S3).The difference in survival time between these two group was not significant, which may due to small sample size.We also assessed the overall survival of patients with EGFR/TP53-mutant lung cancers.Our cohort contained eight patients with co-existing EGFR/TP53-alterations.We compared the mutational signatures between the patients with a good and those with a poor survival outcome.The presence of a co-existing MYC alteration was associated with worse survival in patients with EGFR/TP53-mutant lung ADCs.Moreover, a co-existing AR, FBXW7, or CTNNB1 alteration was associated with better survival in patients with EGFR/TP53-mutant lung ADCs.In our study, a relatively small number of patients with coexisting EGFR/TP53 alterations was identified. Intratumoral heterogeneity presents a major challenge in precision cancer therapy because it can lead to underestimation of the tumor genomic landscape when based on single tumor biopsy samples, and this might contribute to drug resistance and treatment failure [35].All known ClinVar pathogenic mutations were identified in all regions of individual tumors.The predicted pathogenic variants were trunk mutations, with a frequency ranging from 36.36 to 78.95%.We found that the trunk or branch mutations were expressed at a constant level based on the transcriptome data.We found few genes with varying expression levels in different regions of the same sample, and our results differ slightly from those of other study [36], which may be because our selected regions were close in proximity. In summary, we identified genomic aberrations underlying NSCLC in a Taiwanese population.Our study provides putative biomarkers for prognostic prediction in lung cancer.Further research is required to elucidate the functions of these genes and their pathways. • 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": "Discussion",
"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": "The data analyzed in this study were partially provided by the China Medical University Hospital Cancer Registry. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This work was supported by China Medical University Hospital (DMR-109-109). ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https ://doi.org/10.1186/s12931-020-01608 -5. Additional file 1: Table S1.Mutations in five cancer genes in the 72 patients.Table S2.Comparison of driver gene mutations of lung cancer between Taiwanese patients and the Caucasian cohort in TCGA dataset.Table S3.List of trunk mutations of ClinVar pathogenic variants in four paired samples.Table S4.List of trunk and branch mutations of prediction pathogenic variants in four paired samples.Table S5.List of differentially expressed genes with a fold change in expression > 4 in four paired samples.Table S6.Novel mutations in cancer driver genes. YSC and JGC conceived and designed the experiments and wrote the paper.SJT performed computation analyses.YCC and TYL analyzed the data.YTL and JCY performed experiments.HYF collected the specimen and performed clinical review.JGC contributed reagents and materials and interpretation of data.All authors read and approved the final manuscript. The present study was approved by the Institutional Review Board of the China Medical University Hospital (CMUH106-REC1-053). 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/s12931-020-01608 -5. Additional file 1: Table S1.Mutations in five cancer genes in the 72 patients.Table S2.Comparison of driver gene mutations of lung cancer between Taiwanese patients and the Caucasian cohort in TCGA dataset.Table S3.List of trunk mutations of ClinVar pathogenic variants in four paired samples.Table S4.List of trunk and branch mutations of prediction pathogenic variants in four paired samples.Table S5.List of differentially expressed genes with a fold change in expression > 4 in four paired samples.Table S6.Novel mutations in cancer driver genes. ",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "YSC and JGC conceived and designed the experiments and wrote the paper.SJT performed computation analyses.YCC and TYL analyzed the data.YTL and JCY performed experiments.HYF collected the specimen and performed clinical review.JGC contributed reagents and materials and interpretation of data.All authors read and approved the final manuscript. ",
"section_name": "Authors' contributions",
"section_num": null
},
{
"section_content": "The present study was approved by the Institutional Review Board of the China Medical University Hospital (CMUH106-REC1-053). ",
"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
}
] |
10.1371/journal.pone.0234101
|
Notch family members follow stringent requirements for intracellular domain dimerization at sequence-paired sites
|
<jats:p>Notch signaling is essential for multicellular life, regulating core functions such as cellular identity, differentiation, and fate. These processes require highly sensitive systems to avoid going awry, and one such regulatory mechanism is through Notch intracellular domain dimerization. Select Notch target genes contain sequence-paired sites (SPS); motifs in which two Notch transcriptional activation complexes can bind and interact through Notch’s ankyrin domain, resulting in enhanced transcriptional activation. This mechanism has been mostly studied through Notch1, and to date, the abilities of the other Notch family members have been left unexplored. Through the utilization of minimalized, SPS-driven luciferase assays, we were able to test the functional capacity of Notch dimers. Here we show that the Notch 2 and 3 NICDs also exhibit dimerization-induced signaling, following the same stringent requirements as seen with Notch1. Furthermore, our data suggested that Notch4 may also exhibit dimerization-induced signaling, although the amino acids required for Notch4 NICD dimerization appear to be different than those required for Notch 1, 2, and 3 NICD dimerization. Interestingly, we identified a mechanical difference between canonical and cryptic SPSs, leading to differences in their dimerization-induced regulation. Finally, we profiled the Notch family members’ SPS gap distance preferences and found that they all prefer a 16-nucleotide gap, with little room for variation. In summary, this work highlights the potent and highly specific nature of Notch dimerization and refines the scope of this regulatory function.</jats:p>
|
[
{
"section_content": "Notch signaling is a cornerstone of multicellularity and dictates cellular fate and identity.Notch signaling is heavily influenced by microenvironmental cues [1], including adjacent \"sending cells\" which present any of five Notch ligands to up to four Notch receptors expressed on so called \"receiving cells\".Ligand bound and activated Notch receptors undergo a series of proteolytic cleavages which release an active intracellular domain (NICD) [2][3][4].This transcriptionally active fragment translocates to the nucleus to act as a co-transcription factor. Common Notch signaling targets are transcription factors themselves, which have their own broader implications and cascades, culminating in a system that requires a fine-tuned, highly sensitive signaling network.Disruption of Notch signaling, both through over-and under-activation, leads to a variety of developmental abnormalities and cancers [5].Understanding mechanisms behind this precise level of internal control may pave the way for treatments of many of its resulting disorders. The mammalian Notch signaling system consists of four mostly homologous receptors (Notch1-4) which are all activated through this manner.Each NICD molecule can be readily split into three sections; the N-terminus which contains the RBPJ associated module (RAM) domain, the central ankyrin domain, and a variable C-terminus which houses the Pro-Glu-Ser-Thr (PEST) domain used in protein turnover and in some Notch proteins, a transactivation domain (TAD).Through the RAM domain, all Notch proteins bind to the same transcription factor, recombination signal binding protein for immunoglobulin kappa J region (RBPJ, also commonly called CSL, CBF-1/Suppressor of Hairless/Lag-1) [6].Upon NICD binding to RBPJ, a new NICD/RBPJ interface is formed which recruits another co-activator, a member of the Mastermind-like (MAML) family [7].This new tri-protein complex recruits a cascade of other transcriptional machinery to drive transcription of its target genes [8].While each Notch protein contains the conserved RAM and ankyrin domains, their transcriptional activation profiles are not identical and are largely dependent on context within promoter elements [9]. The DNA target sites which the Notch transcriptional activation complex (NTC) binds to have been the subject of thorough analysis.The consensus binding site was originally defined as a \"TP1 element\" with the sequence 5' CGTGGGAAAAT 3' that recruits RBPJ to Notch responsive promoters [10,11].TP1 elements are found in a variety of configurations within promoters.Perhaps most importantly, TP1 elements orientated in a head-to-head directionality and separated by 16 base pairs (bp), also known as sequence-paired sites (SPS), enable cooperative binding of two NICD molecules [12,13].This cooperation results in better repression in the absence of NICD, and enhanced activation in its presence [9,14].Upon modeling of two N1ICD transcriptional cores on a SPS, it was proposed that complex dimerization occurs through the N1ICD ankyrin domain [15] and this was further supported through crystallization of the interface [16].Importantly, theses SPS-driven promoters appear to be dimerdependent.When dimerization was interrupted, N1ICD's transcriptional potential was substantially reduced on promoters containing SPSs [15,16] and could no longer induce T-cell acute lymphoblastic leukemia [17].Together, sequence-paired sites and Notch dimerization appear to be potent regulators of Notch signaling and warrant a closer investigation into their mechanics. In the search for new Notch responsive genes, ChIP-Seq approaches have recently been adopted to identify new SPS sites based on DNA interaction with RBPJ.While the NTC-dimer crystal structure dictates a 16-nucleotide spacer region, ChIP-Seq analysis by Castel, et al. identified a variety of potential SPS-driven genes with spacer regions from 11 to 21 base pairs [18].These possible targets are opposed by in vitro analysis which observed a more limited spacer region of 15 to 17 base pairs [15,19].This discrepancy in spacer length is further complicated due to ChIP-Seq approaches that experimentally identified individual RBPJ binding sites then computationally screened for nearby secondary sites [18][19][20].Screening for secondary sites however is not straightforward since loading of a NTC onto a high-affinity site directly enables cooperativity on cryptic, low affinity sites which may not even resemble traditional RBPJ binding sequences [16,17,19].While the transcriptional outcomes seem to be clear, the mechanisms dictating this SPS-response within promoters and enhancers are not clearly understood. While dimerization-induced signaling of Notch1 has been previously explored, the ability, specifications, and limitations for the other members of the Notch family remain unknown. To compare dimer-dependent signaling of the various NICDs, we generated luciferase reporter constructs driven by either isolated sequence-paired sites from known dimer-dependent promoters or an artificial/optimized SPS site.We observed that all NICDs activate these promoters with varying efficiency.We also observed that Notch1, 2, and 3 functions through dimerization dependent mechanisms, while Notch4 appeared dimer independent.Finally, we compared the optimal gap length within SPS sites and found that all NICD molecules prefer promoters with 16bp between RBPJ binding sites, with little room for deviation.These results should help us to understand how the various NICD molecules interact in cells and potentially diversify Notch signaling outputs in cells that express multiple Notch proteins. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "HEK293T cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Mediatech, Inc.) supplemented with 10% EquaFetal Bovine Serum (FBS) (Atlas Biologicals) and 1x penicillin-streptomycin solution (Mediatech, Inc.).Cells were grown in 10 cm culture plates and subcultured at 70-80% confluency. ",
"section_name": "Cell culture",
"section_num": null
},
{
"section_content": "Protein expression constructs were obtained through the following: FLAG-N1ICD (AddGene #20183), N2ICD (#20184), N3ICD (#20185), and N4ICD (#20186) were all gifted by Raphael Kopan [9] and acquired through AddGene.org.All constructs code for the intracellular domain of the mouse Notch proteins and have a 3xFLAG peptide tag on the N-terminus.The N1ICD-MYC ΔS2184 construct, also a gift from Raphael Kopan [2] (#41730), includes a substantial C-terminal truncation, encoding mouse N1ICD V1744 to S2184 with a MYC tag located at the C-terminus.The NICD coding regions were subcloned into pKH3 (#12555), a gift from Ian Macara [21] to add a C-terminal 3xHA tag.N1ICD (R1974A), N2ICD (R1934A), N3ICD (R1896A), and N4ICD (R1685A) mutants were all created through site-directed mutagenesis of the NICDs based on sequence alignment to identify amino acids (S2 Fig) in mouse NICDs homologous to the human N1CID R1984 site previously shown to be essential for NICD dimerization [16].The empty coding vector pcDNA3.1/MYC-Hiswas obtained from Invitrogen and pCMV-β-Galactosidase was obtained from Clontech/Takara Bio. Transcriptional reporter constructs were obtained or created as the following: Full-length mouse promoters for Hes1 (#41723) and Hes5 (#41724) were a gift from Ryoichiro Kageyama and Raphael Kopan [22].4xTP1 (#41726), a synthetic promoter containing four high-affinity RBPJ binding sites in tandem, was a gift from Raphael Kopan [23].These promoter sequences were designed and cloned into pGL2-Basic (Promega), a luciferase reporter plasmid, which upon promoter activation drives expression of firefly luciferase. ",
"section_name": "Expression and reporter plasmids",
"section_num": null
},
{
"section_content": "To create luciferase reporters that activate specifically upon Notch dimerization, we isolated the sequence-paired sites from the native mouse and human Hes1 and Hes5 genes and cloned these fragments into the pGL3-Basic vector (Promega) which contains a minimal promoter that is incapable of transcriptional initiation without additional enhancer elements.Our synthetic promoters, the 2xTP1 constructs, were designed using the TP1 response element as originally isolated from the Epstein-Barr virus, which contains two high-affinity binding site for RBPJ [10,11].TP1's 'complete' RBPJ consensus sequence (5'-CGTGGGAAAAT-3') and a ubiquitous \"core\" sequence (5'-GTGGGAA-3') were taken from the response element, its secondary site reversed and placed in the complementary strand to result in a head-to-head arrangement, and two nucleotides were inserted into TP1's spacer region to result in a sixteenbase pair, sequence-paired site. These fragments were synthesized as oligonucleotides (Integrated DNA Technologies, IDT) and were designed to be partially complementary so that when annealed, the ends were left overhanging, matching cuts from the restriction enzymes KpnI and SacI (New England Biolabs, NEB).pGL3-Basic was cut with these two enzymes, dephosphorylated with shrimp alkaline phosphatase (NEB), inserts phosphorylated with T4 Polynucleotide Kinase (NEB), and ligated together with T4 DNA Ligase (NEB). A variety of sequence-paired site gap distances were created through blunt-end ligation.PCR primers to mutate Hes1, Hes5, and 2xTP1 were designed to align with the desired base excisions or to include base extensions.Base extensions were designed to keep the gap distance nucleotide composition (G/C vs A/T) approximately consistent with the native promoters'.PCR products were phosphorylated, ligated, and reaction template digested with the restriction enzyme DpnI (NEB).A statistical comparison of the modified constructs' basal activity levels did not indicate the addition or subtraction of any other regulatory elements (data not shown). All constructs were sequenced verified before experimental use.SPS sequence information can be found in S1 Table. ",
"section_name": "Construct creation and mutagenesis",
"section_num": null
},
{
"section_content": "For western blotting analyses, HEK293T cells were plated into 6-well plates at a density of 300,000 cells/well.The following day, cells were transfected with polyethylenimine MW 25,000 (PEI, Polysciences) at a ratio of 5 μg PEI to 1 μg DNA.Wells were transfected with 1000 ng of plasmid DNA for the various FLAG-NICD constructs, allowed to grow for two days, and cells collected and prepared in 1x SDS-page lysis buffer.Western blotting was performed as described previously [24].Membranes were incubated with primary antibodies against FLAG tag (Cell Signaling Technology, #14793) or GAPDH (Santa Cruz Biotechnology, sc-25778) and detected through horseradish peroxidase conjugated α-rabbit antibodies (GE Healthcare Life Sciences, NA934V).Experiments were repeated independently three times, where the figure displayed uses the best representative exposures. ",
"section_name": "Western blotting",
"section_num": null
},
{
"section_content": "For all luciferase assays, HEK293T cells were plated into 24 well plates at a density of 50,000 cells/well where the experimental conditions were treated as triplicates or duplicates.The following day, cells were transfected.When analyzing the full-length promoters, 100 ng of luciferase construct and 10 ng of NICD expression plasmid were used, whereas in experiments of sequence-paired site constructs, 200 ng and 100 ng were used, respectively.In all experimental variations, 10 ng of a β-Galactosidase expression plasmid was used per well to normalize data for transfection efficiency and cell growth/death.To equate amounts of DNA between experimental conditions, the empty coding plasmid pcDNA3.1/MYC-Hiswas utilized.Forty-eight hours post transfection, cells were collected and analyzed as previously described [24].All samples were treated in triplicates, except for the single-base change experiment, which was in duplicates.Independent experiments were performed at least four times. ",
"section_name": "Luciferase assays",
"section_num": null
},
{
"section_content": "Statistical significance was determined through a student's two-tailed t test, comparing twosamples with homoscedastic variance.Significance is determined as ��� is p � 0.001, �� is p � 0.01, and � is p � 0.05. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Notch target genes often have multiple RBPJ binding sites within their promoter sequences and a fraction of these are orientated in head-to-head, paired sites [18,19].This arrangement allows for NTC dimerization through NICD ankyrin domains, resulting in potent transcription of SPS containing genes.The canonical Notch target genes Hes1 and Hes5 have previously shown to be activated by Notch1 in a dimer-dependent manner [16,19].Using luciferase reporter assays, we first sought to confirm if other members of the mammalian Notch family also activate Hes1 and Hes5 in a dimer-dependent manner. HEK293T cells were co-transfected with commercially available luciferase reporter plasmids containing large fragments of the Hes1 or Hes5 promoters, and either wild-type or dimerization incompetent Notch1-4 NICD expression plasmids to observe dimerization dependence of these promoters.In agreement with previous work, the full-length Hes1 (Fig transcriptional activation was significantly less for dimer incompetent N1ICD.Likewise, wildtype N2ICD and N3ICD also displayed activation on both promoters but dimer-dependent activation only on the Hes1 promoter.Indeed, mutant N2ICD and N3ICD dimer incompetent constructs demonstrated a similar ability to activate the Hes5 promoter suggesting the N2ICD and N3ICDs were functioning in a dimer-independent manner on the Hes5 promoter.Finally, N4ICD activated the Hes1 promoter to a similar degree as the other NICDs but was significantly weaker on the Hes5 promoter compared to the other NICDs.Moreover, N4ICD failed to demonstrate dimer dependence on either the Hes1 or Hes5 promoters.In agreement with previous observations [15], western blot analysis of the various NICDs demonstrated that the decreased transcriptional activation of the dimer incompetent ankyrin mutant NICD molecules was not associated with decreased protein expression of these mutant NICDs (Fig 1C).Moreover, mutation of NICD ankyrin domains did not impinge the basal transcriptional activity of NICD molecules since WT and ankyrin mutant NICDs exhibited nearly identical transcriptional activity on the non-dimerizing 4xTP1 promoter (Fig 1D). Collectively, these results largely supported the previously reported dimer-dependent nature of the Hes1 and Hes5 promoters.However, we also noted several weaknesses with these experiments.In particular, we noticed significant background with modest activation of the Hes1 promoter and reduced dimer dependence for N2-4ICD on the Hes5 promoter.Based on these weaknesses, we more closely examined these promoter sequences (S1 Fig) .We searched for high affinity binding sites that were defined by the RBPJ consensus sequence TGTGGGAA, and low affinity sites that were defined as having up to two nucleotide differences compared to the high-affinity sites.We found a multitude of possible low affinity RBPJ binding sites in addition to the high-affinity SPS sites previously described [9].Based on this result, we hypothesized that the overall promoter complexity and number of potential low affinity RBPJ binding sites within these promoter sequences might have been responsible for the high background, low relative activation, and dimer independence observed in our experiments.Moreover, beyond Notch signaling, Hes genes are also controlled by a variety of other transcription factor families [25,26], including self-regulation [27,28], suggesting that these relatively large promoter fragments might have been responding to both Notch specific and non-Notch transcriptional activity.Collectively, these observations prompted us to perform further experiments on the Hes1 and Hes5 promoters with the goal of refining these promoters into more specific tools to monitor dimer-dependent NICD activation. ",
"section_name": "Activation of Notch target genes containing sequence-paired sites requires ankyrin-dependent dimerization",
"section_num": null
},
{
"section_content": "Our results in Fig 1 show that the Hes5 promoter demonstrated both stronger activation than the Hes1 promoter and N1ICD dimer dependence.We therefore rationalized that isolation of the Hes5 SPS site should yield a promoter element that would specifically and robustly respond to NICD dimerization.We isolated the mouse and human Hes5 SPS elements containing two head-to-head orientated RBPJ binding sites separated by a 16-nucleotide gap, and the 4 to 5 surrounding nucleotides (Fig 2A ) as previously described [16] and cloned these sequences into a luciferase reporter vector containing a minimal promoter that was incapable of transcriptional initiation without additional enhance elements.Interestingly, this cloned region includes one canonical RBPJ binding site and a partnered \"cryptic site\" which doesn't match standard RBPJ binding sequences, but instead was hypothesized to form the partner site for the high-affinity site [16].In addition, there are two nucleotide differences between the mouse and human genes in this region (Fig 2A ), one of which is inside the cryptic RBPJ binding site.As shown in Fig 2B , we found that both mouse and human isolated Hes5 SPS promoters were indeed responsive to N1ICD, however, neither of these isolated elements appeared to be dependent on NICD dimerization since they were equivalently activated by WT and dimerincompetent versions of N1ICD.In addition, the isolated Hes5 SPS responded to Notch activation almost exactly the same as to promoters with RBPJ binding sites in a non-dimerizing head-to-tail orientation (i.e.2xTP1(H-T) and 4xTP1(H-T) constructs).Moreover, N1ICD activated the Hes5 SPS construct less than the 2xTP1 construct despite these promoters both containing two RBPJ binding sites.Interestingly, N4ICD was unable to activate transcription from the isolated Hes5 SPS sites despite successfully activating the full length Hes5 promoter in Fig 1 .In stark contrast to this result, the full-length Hes5 promoter did display dimer dependence to N1ICD (Fig 1B).This difference between the full-length and SPS versions of the Hes5 promoter suggested that the Hes5 promoter might have been functioning differently than previously thought.Given the weakness of the cryptic RBPJ binding site, the presence of several other potential RBPJ binding sites within the full-length promoter, and the dimer dependence of the full-length Hes5 promoter, we hypothesized that the high affinity RBPJ binding site might be making a long-distance interaction with another high-affinity site within the promoter.This possibility would allow cooperation between previously thought independent NTCs within gene promoters and enhancers and perhaps explain the transcriptional differences between full-length and SPS versions of the Hes5 promoter.To test this possibility, we designed a series of Hes5 SPS mutants based on the helical nature of DNA.Since a DNA helix completes approximately one rotation every 10 bp, we hypothesized that an insertion or deletion of five extra base pairs would place the secondary NTC on the opposite side of the DNA, breaking any cooperativity.Further, an addition of an extra five nucleotides may restore cooperativity, despite the NTC being further away, due to a long range NTC dimerization.However, no obvious cooperative binding, or loss thereof, was observed in these constructs (Fig 2C). Collectively, these results indicated that the Hes5 SPS does not function in a dimer-dependent manner in cells and instead behaves as a monomeric RBPJ binding site.Due to this unexpected complication, to further explore SPS capabilities we moved on to the Hes1 promoter, which contains a more canonical SPS site. ",
"section_name": "The isolated Hes5 sequence-paired site does not respond to dimerization",
"section_num": null
},
{
"section_content": "Hes1 is another Notch target gene which demonstrates dimer-dependence.Its promoter contains a single canonical RBPJ binding site and secondary site 16 nucleotides away in the reverse orientation (Fig 1A).This secondary site is slightly non-consensus, but only displays a minor decrease in RBPJ affinity compared to the high affinity consensus site [19,29].Therefore, we took the same reductionist approach used with Hes5 and isolated the SPS element out of the Hes1 promoter. Since the human and mouse sequences in this promoter region are identical, a generic Hes1(SPS) construct was cloned into a minimal promoter luciferase vector to quantitatively monitor its activation by Notch signaling (Fig 3A ,Top).As before, transfections of this construct into HEK293T cells, along with WT or ankyrin mutated NICDs, allowed us to analyze dimer dependence of the Hes1 SPS element.The Hes1(SPS) construct responded to NICD activation (Fig 3A ,Bottom) and interestingly, the isolated Hes1 SPS demonstrated approximately 5 times greater sensitivity to N1ICD compared to the full-length Hes1 promoter, most likely due to reduced background of the isolated SPS construct.Both N1ICD and N2ICD ankyrin mutants showed a significant decrease in activation compared to their wild-type counterparts.In comparison, N4ICD was again dimer independent, matching trends observed on the counterpart full-length promoter (Fig 1A).Finally, N3ICD only weakly activated the isolated Hes1 SPS.Indeed, compared to the full-length Hes1 promoter, N3ICD showed no increased activity on the isolated Hes1 SPS.Moreover, N3ICD did not demonstrate dimer dependence on the isolated Hes1 SPS but did demonstrate dimer dependence on the fulllength Hes1 promoter (Fig 1A).This is potentially unsurprising since Notch3 has been shown to synergistically utilize other nearby transcription factors for its own signaling responses [9], which would be lacking in this minimalized promoter. With a properly responding SPS-driven promoter, we again wanted to determine if longrange interactions were possible between RBPJ binding sites.We again followed the same logic employed when mutating the Hes5 SPS and we created gap distances in steps of five nucleotides to take advantage of the periodicity of the DNA helix.We found that the wild-type, 16-nucleodtide gap, was the only distance all NICDs were capable of cooperatively binding and eliciting activation on (Fig 3B).This observation further limits the options of dimerization dependent signaling since independent NTC sites within this promoter are unlikely to cooperate over long distances by methods of kinking, looping, or untwisting the gap DNA. ",
"section_name": "The Hes1 SPS acts through traditional NICD dimerization mechanisms",
"section_num": null
},
{
"section_content": "Having found that the SPS element isolated from the Hes1 promoter functions in a dimerdependent manner, we next sought to optimize an SPS-driven luciferase construct as a tool to specifically study the transcriptional activity of NICD dimers.To accomplish this, we constructed a synthetic SPS site that contained two high-affinity RBPJ binding sites in a head-tohead configuration separated by 16 bp.We utilized the complete TP1 consensus sequence (5'-CGTGGGAAAAT-3') originally described by Meitinger et al., [10] thus forming the 2xTP1(SPS)-Complete construct.RBPJ shows high affinity towards this site and multiple copies of this RBPJ binding site have previously been arranged in a head-to-tail orientation to measure the activation of Notch signaling [11,30,31].These sequences however have not previously been orientated in a head-to-head orientation to measure transcriptional activation by dimerized NICD molecules. As shown in Fig 4A, the 2xTP1(SPS)-Complete construct successfully responded to Notch signaling.Unexpectedly however, dimer-incompetent N1ICD mutants increased activation even better than wild-type proteins indicating this promoter was not dimer-dependent.We hypothesized that since the last three nucleotides in the consensus sequence were not necessary for RBPJ-DNA interactions [10], this effectively resulted in a 22 nucleotide gap distance between RBPJ binding sites, and therefore the promoter lost its dimerization dependence.In response to this possibility, we created another SPS construct, this time with a core, essential sequence for RBPJ responsiveness (5'-GTGGGAA-3') [10,32] separated by 16 bp.We named this new construct the 2xTP1(SPS)-Core construct.Cloning of this fragment resulted in a 'T' on the 5' side of each core sequence.This addition coincidentally matches the RBPJ consensus sequence found in Hes1/5, though this position within the consensus is variable [33].As shown in Fig 4B, wild-type N1, N2, and N3ICD all strongly activated this promoter while the corresponding dimer-incompetent ankyrin domain mutants demonstrated significantly reduced transcriptional activity.N4ICD also activated the promoter, but remained dimerization independent, which has persisted across all SPS promoter variations tested.Nonetheless, the 2xTP1(SPS)-Core construct appeared to be an optimized promoter for evaluating dimer-dependent Notch signaling. ",
"section_name": "Establishment of a high activity, NICD dimer-specific reporter construct",
"section_num": null
},
{
"section_content": "The results in Fig 4A revealed an interesting phenomenon wherein non-dimerizing ankyrin mutant NICDs performed better than their WT counterparts on the 2xTP1(SPS)-Complete construct which had a slightly longer gap than the 16 bp preferred by NICD molecules.This observation suggested that two RBPJ binding sites gapped slightly more or less than 16 bp within a promoter might actually suppress promoter responsiveness to NICD dimer-dependent Notch signaling and favor NICD dimer-independent Notch signaling, a phenomenon which has not been previously described.To test this hypothesis, we compared WT and ankyrin mutant N1ICD transcriptional activation from the Hes1(SPS) and 2xTP1(SPS)-Core constructs which we modified to contain head-to-head RBPJ binding sites separated by 11, 16, or 21 bp.As shown in Fig 5A and5B, WT N1ICD strongly activated reporter transcription on the 16 bp gap promoters and was significantly weaker on the 11 bp and 21 bp gap promoters.Moreover, the ankyrin mutant N1ICD also performed as expected, showing no synergistic activity across the various gap distances.Importantly however, the ankyrin mutant N1ICD slightly outperformed the WT N1ICD on the 11 bp and 21 bp gap promoters (Fig 5A and5B right panels).This observation suggested that head-to-head orientated RBPJ binding sites with non-optimal gap widths are more likely to be activated by non-dimerizing NICD molecules.Whether or not there is a condition that actively manipulates NICD dimerization is currently unknown. ",
"section_name": "Non-optimal SPS sites select against transcriptional activation by NICD dimers",
"section_num": null
},
{
"section_content": "Having established a highly active and NICD dimer-specific promoter, we set out to compare the promoter specificity of the various NICD molecules.To accomplish this, we established a series of 2xTP1(SPS)-Core promoters with modified SPS gap distances ranging from 11 to 21 bp in one bp increments.Combinations of each NICD protein coupled with each spacer variation were then transfected into HEK293T cells and assayed for their transcriptional activity.Our rational for this approach was the previous finding that ChIP-Seq screening identified SPS-driven genes with various gap distances, from 11-21 base spacers [18].Our data in Fig 3 did not support the idea that gap distances greater or less than 16 bp (in 5 bp increments) can support dimer-dependent transcription on the Hes1 promoter.Nonetheless, we wanted to use our optimized promoter to investigate the possibility that smaller variation in gap distances may be tolerated during NICD dimerization, or that different Notch family members may exhibit slightly different preferences in SPS gap width.As shown in Fig 6, all mouse NICDs have a strong preference for the SPS sites with 16 bp gaps, with little room for variation.For N1ICD, there was some flexibility observed with 15 and 17 bp spacers, though anything outside of this range did not demonstrate cooperative signaling (Fig 6A).In contrast, N2ICD showed even more specificity, where there was only slight cooperativity at the 15 bp gap, and none observed at 17 (Fig 6B).N3ICD showed cooperativity at 16 bases (Fig 6C ), and N4ICD at 16 and 17 bases (Fig 6D ), however it's difficult to make a judgement call about their flexibility due to their low activation and their apparent dimerization independent signaling.Also, it's worth noting is that N4ICD preferred a 16 bp gap suggesting that N4ICD was functioning as a dimer which is in conflict with our earlier observations that N4ICD functioned dimer-independently.Moreover, N4ICD demonstrated slight inhibition on promoters with 11 or 14 bp gaps, which was similarly observed in the Hes5 (Fig 2C ) and Hes1 (Fig 3B) SPS promoter constructs.This might indicate that N4ICD proteins generally act as transcription inhibitors on monomeric NTC sites, or perhaps occupy RBPJ binding sites and prevent other NICDs from binding, though we did not explore these possibilities.Further we also observed a slight increase in activation of the 12 or 13 bp gap constructs for all four NICD proteins, which could be attributed to some form of cooperativity, though we did not explore this thread any further.Finally, as with the Hes5(SPS) and Hes1(SPS), we extended the gap distances of the 2xTP1 (SPS)-Core out to 36 nucleotides and again observe little to no long-range cooperativity. ",
"section_name": "A restrictive spacer range dictates the signaling capabilities of NICD dimerization",
"section_num": null
},
{
"section_content": "Notch signaling is an important cellular communication mechanism that is required for multicellular organisms.Ongoing research continues to reveal how Notch functions and how Notch signaling is integrated into many facets of cell biology.Despite our growing understanding of Notch function, however, there are basic questions about Notch signaling that remains to be addressed.In this study, we sought to address some of these basic questions about Notch signaling that have been overlooked in the quest to dig into the deeper questions of Notch function.In particular, we felt that a head-to-head transcriptional comparison of the full-length Notch NICD molecules on a variety of promoter elements should be performed.Most of what we know about Notch dimerization, including how the proteins interact and on which promoters, comes from studies with Notch1.While the four mammalian Notch proteins are mostly homologous within the N-terminal and ankyrin domains, there are substantial differences within the C-terminal regions.Notably, this domain is absent in much of the field's previous characterization work.Indeed, molecular modeling [15] and crystallization [16] were performed with just the ankyrin domain, and these studies laid the original groundwork for the NTC's spatial conformations, interacting amino acids, and DNA preferences.Similarly, further in vitro work with EMSAs and FRET assays supported NTC cooperative loading and SPS gap preferences, though these were performed using N1ICDs with just the N-terminal RAM and ankyrin domains [15,16,19].Importantly, the C-terminus has known transcriptional effects [34], regulatory capacity [35], and contains a multimerization site [36].Therefore, it was important to ensure 1) the presence of these domains don't interrupt dimerization responses in a cellular context, and 2) if the lessons learned by studying Notch1 can be broadly applied to the other mammalian Notch proteins. To address these concerns, we first revisited full-length, Notch-responsive promoters, which contain canonical sequence-paired sites to drive transcription.While these promoters did require NICD dimerization for full activation and therefore allowed for preliminary conclusions, our data collection suffered from low activation levels (Hes1) or high experimental variability (Hes5).This is a logical byproduct from using full-length promoters since these genes are regulated through mechanisms beyond just Notch signaling and overexpression of constitutively active NICDs would have wide, overarching, signaling outcomes.For example, as in the case of Hes1, this gene is understood to be self-regulated, resulting in its oscillatory nature [37].For our purposes, this implies that endogenous Hes1 may be activated by NICD overexpression and circles back to our Hes1-luciferase promoter to negatively-regulate it, resulting in its poor overall activation.For reasons like this, we sought to create minimalized promoters to more specifically monitor dimerization-induced signaling. Since synthetic promoters with tandemly arranged RBPJ sites had been previously used to monitor Notch activity (e.g.4xCSL-Luc, [23]), we reasoned that the SPS alone should also be sufficient for Notch activation.Therefore, we isolated the known sequence-paired sites out of Hes1 and Hes5 and created the Hes1/5(SPS)-luciferase vectors.Since the reverse sites within Hes1 and 5 do not perfectly match the canonical RBPJ binding site, we also crafted the synthetic 2xTP1(SPS)-Core promoter to make a \"perfect\" SPS element.Upon testing we found that the Hes1(SPS) and the 2xTP1(SPS)-Core promoters operated as expected in that they were robustly activated by N1ICD and inefficiently activated by dimer-incompetent NICD ankyrin mutants.Surprisingly however, our results did not indicate that the isolated Hes5 SPS site functions in a dimer-dependent manner, although the full-length Hes5 promoter was dimer-dependent.We return to this point later in this discussion. Having established two reporters with high activity and specificity for dimerized NICD signaling we were able to specifically ask questions about dimerization of full-length NICD proteins in cells.We found that full-length N1ICD and N2ICD activated sequence-paired sites in a dimerization-dependent mechanism.In contrast, N3ICD's activation of these minimal promoters was poor, which may be partially attributed to its lack of a TAD domain in its C-terminal [34] or usage of other transcription factors for its activation [9].It also differed in its dimer-dependence; where it was not dimer-dependent on the Hes1(SPS) but was on the 2xTP1(SPS)-Core.This implies there is some difference located within these two minimalized constructs that can affect dimerization outcomes.The primary source of variation can be found within the 16 bp gap between RBPJ binding sites.The original crystallography studies found that the Hes1 gap distance DNA must go through substantial untwisting to bring the ankyrin domains into contact with each other for dimerization [16].Gap composition of the 2xTP1(SPS)-Core construct has even higher G/C content than Hes1's, so while N1ICD and N2ICD can utilize variable gap compositions, perhaps N3ICD utilizes a more inflexible gap sequence.Whether or not this would enable differential transcriptional activity of Notch remains to be tested.In contrast, however, N4ICD did not appear to have dimer-dependent activation on the Hes1(SPS) or 2xTP1(SPS)-Core constructs despite still displaying a preference for a 16 bp gap between RBPJ binding sites on these promoters.Based on these results, we believe that the single R1685A mutation (which ablates dimerization of the other NICDs) on N4ICD was insufficient to abolish its dimerization activity suggesting that perhaps N4ICD has an alternative dimerization interface.Indeed, crystallography work on human N1ICD homodimers indicated two other amino acids (K1945 and E1949) beyond the R1984 that are involved in ankyrin-mediated dimerization [16].Interestingly the N4ICD is the only NICD with an \"R\" located at the K1945 site, suggesting this position may be especially important for N4ICD dimerization (S2 Fig). In addition to comparing the activation parameters of the various NICDs on these optimized promoters, we were also curious about the standard 16-nucleotide gap distance established by the N1ICD containing tripartite complex.Two hypotheses presented themselves to us.First, given the contortion of DNA evident in the crystalized N1ICD trimeric complex, we hypothesized that RBPJ sites outside of the optimal 16 bp gap might be utilized through further contortions of DNA between RBPJ binding sites.Second, we further hypothesized that given the variable sequences and sizes of the NICD proteins, different NICDs might have preferences for slightly longer or shorter gap distances between RBPJ binding sites.To test the first hypothesis, we created SPS sites with variable gap distances in 5 bp increments to take advantage of the periodicity of helical DNA.We found that none of these long-range alternative gap distances, for any of the NICDs, had significantly enhanced signaling above monomeric signaling.This indicated that long-range interactions between NTC complexes are unlikely to occur.Our analysis however only extended to measuring 36 bp gaps and therefore, longer-range interactions between RBPJ binding sites cannot be ruled out.To address the second hypothesis (that different sized NICDs might have subtle differences in SPS preference), we compared NICD activity on SPS sites with 11-21 bp gap distances in one bp increments.Similar to previous results [16,19,38], we found that all full-length NICDs also prefer a 16 bp gap distance, with little room for variation.Based on this result, we conclude that the C-terminal regions of NICDs do not appear to change the SPS dimensions preferred by NICDs nor impact NICD dimerization.It should also be stated, however, that deletion of the N1ICD C-terminal tail (ΔS2184) which contains the trans-activation domain (TAD) significantly reduced transcriptional activity (S3 Fig) .Minor differences in the NICDs however were observed.For example, N2ICD appeared slightly more restrictive when choosing SPS sites than Notch1, since N2ICD activated 15/16 bp spacers but not the 17 bp gap.This poses an interesting possibility where N1ICD might dimer-dependently activate CUL1 or TXN2, which have SPSs with a 17-nucleotide gap distance [16,18], whereas binding of N2ICD on this promoter would only cause monomeric levels of transcription, though this idea was left untested.While N3ICD and N4ICD had questionable dimerization activities, they did seem to have cooperative binding and higher activity on the 16/17 bp gap SPSs.Making firm conclusions about their preferences is difficult however due to their low overall activity and high variability.Together, these experiments highlight the stringent requirements for dimerization-induced transcription. Our results have also shed some light on the nature of NICD dimerization.First, there has been some debate as to the order of events leading up to NICD activated transcription.It is currently unclear whether individual NICD molecules first bind to RBPJ/DNA then form dimers, or alternatively if two NICDs first form a dimer, then bind to RBPJ/DNA.In Figs 4 and5, we demonstrated that dimer-incompetent N1ICD performed slightly better than WT N1ICD on SPS elements with non-optimal 11 or 21 bp gaps.This suggests that the attempt to dimerize may impede NICD molecules from binding to these sites and might be a clue as to the mechanics of NICD function.Our result indicates that non-optimal SPS elements discourage NICD dimer formation and we believe this is evidence that NICD molecules are pre-forming dimers before binding to RBPJ/DNA.Whether or not this has an actual impact on how promoters with non-optimal SPS sites are utilized by Notch signaling remains to be seen but as shown by Castel et al., [18], several Notch responsive promoters with non-optimal SPS elements have been identified.Another outstanding question about NICD dimerization is whether or not NICD molecules can engage in heterodimerization.Given the conservation of sequence in the ankyrin domains and the importance of ankyrin domains for NICD dimerization it has been hypothesized that NICD heterodimers may exist.While further research on this topic is certainly warranted, our results in Fig 6 showing similar SPS element preferences and in Fig 4 showing ChIP recovery of N1/N4 complexes suggests that heterodimers between N1ICD and N4ICD can form in transfected 293T cells and therefore possibly under more physiological conditions.Here again, the biological implications of this observation are unknown but given the strong differences in transcriptional activation between N1ICD and N4ICD, we hypothesize that N1/N4ICD heterodimers would have intermediate activity compared to N1ICD or N4ICD homodimers.Thus, heterodimerization of NICD molecules may offer a new mechanism to regulate outputs from Notch signaling. Finally, our minimized Hes5(SPS) promoter was not sufficient to elicit dimerization-dependent activation.The Hes5 promoter does not contain a canonical sequence-paired site and instead has been described as 'cryptic' [16], with a standard forward RBPJ binding site but an abnormal reverse site.When arranged as a palindrome in a SPS, this abnormal reverse site does not support dimerization, yet it facilitated dimerization when paired with a strong RBPJ binding site [19].Work with EMSAs of this cryptic SPS showed that N1ICD homodimers can form, and dimerization-dependent activation through this site was supported through luciferase assays [16], but distinctly, these luciferase assays were still performed with the full-length promoter.In our analyses we isolated out this SPS, which should be sufficient for dimerization, yet this construct did not demonstrate dimerization-dependent activation. This inconsistency poses two thought-provoking problems.First, and as originally described by Severson et al. [19], if cryptic sequence-paired sites are capable of forming NICD dimers, then searching for SPSs by 'sequence-gazing' becomes far more difficult.For example, previous ChIP work isolated out RBPJ-bound DNA targets, and these sequences were screened for nearby RBPJ binding motifs located in tandem [18].The issue here lies in the partner sequence, wherein any non-conforming RBPJ sequences would be missed through a simple screening approach.To further identify other cryptic sequence-paired sites, like those in Hes5 or pTα [16,17], we propose that a logical course of action is to perform ChIP-Seq on a doubleselected pool of DNA that specifically identifies two dimerizing NICD partners.The second problem concerns the activation of Hes5 through its SPS.While it appears that NTC complexes dimerize on this promoter segment in vitro, do they still form in living cells and if so, what's the missing link for transcriptional activation?Other than the cryptic RBPJ binding site, the Hes5(SPS) construct is nearly identical to the Hes1(SPS) and 2xTP1(SPS)-Core constructs which respond as expected.Since the full-length Hes5 promoter is apparently dimerresponsive and the other minimalized SPS constructs are sufficient for activation, we predict that there are other promoter elements involved which enable these low-affinity dimers to form and signal in a cellular context.Comparing the promoters and enhancers of multiple, cryptic, SPS genes may identify other sequence motifs in common and identify signaling or regulatory pathways involved. In conclusion, since much of the work on NICD dimerization has been performed studying C-terminally truncated N1ICD, we felt it was important to 1.) examine full-length NICD molecules to insure the C-terminal domain does not affect NICD promoter preference, and 2.) compare promoter preferences of the other NICD molecules, which have been largely overlooked.In so doing, we confirmed that while the C-terminal domain of the various NICDs has a transcriptional role, this domain does not appear to play a role in promoter preference.In addition, we also found that all the mammalian NICDs have remarkably similar SPS gap length preferences with only minor (+/-one bp) flexibility.Overall, these results both support previous work but also help fill in missing gaps in our understanding of Notch transcriptional activity. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "This work was supported by funding from the National Institute of General Medical Sciences (https://www.nigms.nih.gov) to A. Albig (2R15GM102852-02 and 1R15GM134501-01) and from NIGMS grants P20GM103408 and P20GM109095. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "All relevant data are within the manuscript and its Supporting Information files. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Supporting information",
"section_num": null
}
] |
10.1186/1741-7007-9-83
|
Notch1 binds and induces degradation of Snail in hepatocellular carcinoma
|
<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>Hepatocellular carcinoma (HCC) is a common, highly invasive malignant tumor associated with a high mortality rate. We previously reported that the aberrant expression of Snail via activation of reactive oxygen species contributes to the invasive property of HCC, in part by downregulation of E-cadherin through both transcriptional repression and epigenetic modification of the E-cadherin promoter. Having demonstrated the ability of Snail to bind and recruit histone deacetylase 1 and DNA methyltransferase 1 in this context, we set out to look for other interactions that could affect its ability to promote oncogenic transformation and cancer cell invasion.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>Using cells that stably expressed Snail, we characterized Snail protein interactors by tandem affinity purification and mass spectrometry. Immunoprecipitation and subcellular colocalization studies were performed to confirm our identification of the Notch1 intracellular domain (NICD) as a novel Snail-binding partner. NICD interaction with Snail was found to induce ubiquitination and MDM2-dependent degradation of Snail. Interestingly, NICD inhibited Snail-dependent invasive properties in both HCC cells and mouse embryonic fibroblasts.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p>Our study demonstrates that NICD can oppose Snail-dependent HCC cell invasion by binding and inducing proteolytic degradation of Snail. Although Notch signaling and Snail are both widely considered tumor-promoting factors, our findings indicate that the individual oncogenic contribution of Notch1 and Snail in malignant systems should be interpreted carefully, particularly when they are conjointly expressed.</jats:p> </jats:sec>
|
[
{
"section_content": "Hepatocellular carcinoma (HCC) is a common, highly invasive malignant tumor associated with a high mortality rate [1,2].The resistance of HCC to existing antineoplastic agents and the limited effectiveness of chemotherapies due to underlying liver disease contribute to the poor prognosis for patients with HCC [3].Although surgical resection is the preferred standard of care for patients with HCC, few patients are suitable candidates for this treatment and recurrence is common even after radical curative resection [3,4].Given the inadequate impact of conventional therapies and the rising incidence of HCC, elucidation of the oncogenic mechanisms underlying HCC development is critical for identifying potential therapeutic targets or modalities. Snail is a well-known zinc finger (ZF) transcriptional repressor responsible for epithelial-to-mesenchymal transitions (EMTs) and metastasis in several cancers [5,6].We previously reported that upregulation of human SNAI1 (Snail) expression by reactive oxygen species contributes to the invasive nature of HCC, in part by inducing the expression of matrix metalloproteinases and downregulating E-cadherin expression through both transcriptional repression and epigenetic modification of the E-cadherin promoter [7].Other recent reports have revealed that Snail induces broad epigenetic modifications of target genes by interacting with tumor-associated proteins (including HDAC1, DNMT1 and p53) [7][8][9].Taken together, the ability of Snail to promote oncogenic transformation and cancer cell invasion is likely mediated by its interactions with other proteins in addition to its transcriptional activity. The Notch signaling pathway regulates embryonic cell determination and differentiation as well as postnatal development [10,11].Although alterations in the Notch pathway are associated with malignant processes, there is also evidence that supports a tumor-suppressive role for Notch signals [12].Activation of the Notch pathway is initiated through juxtacrine ligand-receptor interactions and the proteolytic cleavage of Notch1 by γ-secretase, which liberates the Notch1 intracellular domain (NICD) from the membrane, allowing NICD to translocate to the nucleus [10].Nuclear NICD then associates with CSL (CBF1/RBPJk in vertebrates, Suppressor of hairless in Drosophila, Lag-1 in Caenorhabditis elegans) transcriptional factors to inhibit CSL transcriptional repression of Notch target genes [10].Of note, though both NICD and Snail proteins are known to play a central role in cancer cell growth, invasion and metastasis, Notch signaling is also capable of inhibiting HCC tumor growth through the induction of cell-cycle arrest and apoptosis [5,[13][14][15]. In this study, we have identified NICD as a novel Snail binding partner by using tandem affinity purification and mass spectrometry (MS/MS) in HCC cells.Using HCC cell lines and mouse embryonic fibroblasts (MEFs), we demonstrate that NICD can induce Snail degradation and impede Snail-dependent cell invasion.Although Snail is known to be degraded by the β-TrCP1 and FBXL14 E3 ubiquitin ligases, our data indicate that NICD-mediated Snail degradation may instead be dependent on MDM2 [16,17]. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To elucidate regulatory factors of Snail, we sought to identify its binding partners by performing MS/MS (Figures 1A and 1B and Additional file 1).We identified Notch1 as a Snail-associated protein (Figures 1B and1C) and confirmed an endogenous Snail and Notch1 interaction by performing coimmunoprecipitation and immunoblot assays (Figure 1D).In both analyses, the size of the detected Notch1 protein was approximately between 100 and 150 kDa, suggesting that Snail binds to the proteolytically cleaved intracellular domain of Notch1, known as NICD.Using the Duolink II assay (Olink Bioscience, Uppsala, Sweden), we have shown that endogenous Snail and Notch1 proteins interact in multiple cancer lines (Figure 1E and Additional file 2, with each red dot in the images representing a fluorescent signal from a Snail-Notch1 interaction).Colocalization of NICD and Snail in nuclear foci by immunofluorescence corroborates the interaction of the two proteins (Figure 1F). The ANK domain of NICD and the zinc finger domain of Snail are required for NICD and Snail interaction NICD contains a RBP-J -associated module (RAM) domain, seven ankyrin/cdc10 repeats (ANK) domain, two nuclear localization signals, a transcriptional transactivation domain (TAD), a polyglutamine tract (OPA) domain and a proline-, glutamic acid-, serine-and threonine-rich (PEST) domain [10] (Figure 2A).Snail contains a highly conserved C-terminal region, a ZF domain and a divergent N-terminal (SNAG) domain (Figure 2C) [5].We mapped interacting domains of NICD and Snail by coimmunoprecipitation analysis using deletion mutants of both proteins and found that the Snail ZF domain and the NICD ANK domain were responsible for the interaction between the two molecules (Figures 2B and2D).To exclude the possibility that altered cellular localization was responsible for the inability of the Snail-ΔZF mutant to bind NICD, we performed glutathione S-transferase (GST) pull-down assays using GST-Snail wild-type (WT), ΔZF and ZF mutants, which showed that only the Snail-ΔZF mutant did not interact with NICD (Figure 2E). ",
"section_name": "NICD is a novel binding partner of Snail",
"section_num": null
},
{
"section_content": "To clarify the role of the interaction between NICD and Snail, we transfected Hep3B cells with hemagglutinin (HA)-Snail and Myc-NICD constructs.Interestingly, we observed that the HA-Snail protein was dramatically decreased in a dose-dependent manner in the presence of Myc-NICD (Figures 3A and3C).Under the same experimental conditions in which NICD and Snail were coexpressed, there was no change in the level of Snail mRNA in comparison with cells that expressed HA-Snail alone (Figures 3B and3D).Furthermore, we performed additional experiments using endogenous Snail protein-upregulated conditions.In our previous study, reactive oxygen species (ROS) stress such as H 2 O 2 treatment-upregulated Snail mRNA and protein expression [7].In the H 2 O 2 -treated cells, Myc-NICD downregulated the Snail protein level, not the mRNA level, although in normal conditions Myc-NICD upregulated Snail mRNA expression (Figures 3E and3F).Collectively, these results suggest that NICD may decrease Snail expression through a posttranscriptional mechanism. ",
"section_name": "NICD downregulates Snail protein",
"section_num": null
},
{
"section_content": "NICD-induced decrease of Snail protein was inhibited by treatment with the proteasome inhibitor MG132, suggesting that NICD promotes Snail degradation (Fig- ure 4A).Treatment of Snail and NICD cotransfected cells with cycloheximide, an inhibitor of protein biosynthesis, revealed that the protein half-life of Snail is shortened in the presence of NICD (Figure 4B).As Snail is known to be tagged by ubiquitin for proteolytic degradation, we sought to determine whether NICD could induce Snail ubiquitination by examining ubiquitinated Snail in cells cotransfected with Flag-Snail and Myc-NICD WT or Myc-NICD ΔANK mutant (Mut).Because NICD ΔANK Mut does not interact with the Snail protein, we hypothesized that Snail would not be ubiquitinated in its presence.In agreement with this hypothesis, ubiquitinated Flag-Snail was detected only in Flag-Snail and Myc-NICD WT cotransfected cells and not in Flag- Snail and ΔANK Mut transfected cells (Figure 4C).These data support a role for NICD in regulating ubiquitin-dependent Snail degradation. Coimmunoprecipitation experiments showed that Myc-NICD can interact with MDM2 (Figure 4D).In a previous report, we found that Snail may also bind MDM2 (Figure 4D) [18].These data support the notion that NICD induces degradation of the Snail protein via MDM2-mediated ubiquitination because NICD has no known intrinsic E3 ligase function.Indeed, in Hep3B cells cotransfected with Myc-NICD and MDM2 siRNA, NICD-induced Snail degradation was decreased compared to control siRNA-transfected cells (Figure 4E).Glycogen synthase kinase 3β (GSK-3β) is known to induce Snail phosphorylation and degradation through the E3 ubiquitin ligase β-TrCP1 [17].To determine whether NICD-induced Snail degradation is reliant on GSK-3β/β-TrCP1, we analyzed the protein level of the Snail 2SA mutant, which has two point mutations in the β-TrcP motif, in cells cotransfected with Snail 2SA mutant and NICD.We found that both the Snail WT and 2SA mutant proteins were uniformly degraded by NICD, implying that NICD-induced Snail degradation is a β-TrcP-independent phenomenon (Figure 4F). ",
"section_name": "NICD induces degradation of the Snail protein by ubiquitination",
"section_num": null
},
{
"section_content": "To determine whether NICD could regulate endogenous levels of Snail protein, we treated Hep3B and Huh7 cells with Notch1 siRNA to silence NICD expression.In Notch1 siRNA-transfected cells, endogenous Snail protein levels were elevated relative to control siRNA-transfected cells (Figure 5A).We confirmed these results using a murine stem cell virus (MSCV) retroviral system to express NICD (MSCV-NICD) in MEFs and Hep3B cells.In both cell types, NICD expression led to decreased levels of endogenous Snail protein but upregulation of p21, an established Notch target gene (Figure 5B).Correspondingly, expression of Jagged1, an activating ligand of Notch signaling, repressed Snail protein levels only in the absence of DAPT (N-[N-(3, 5- difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester), a γ-secretase inhibitor (Figure 5C).Taken together, our results thus far show that physiological activation of the Notch pathway or NICD expression is necessary and sufficient for Snail degradation. ",
"section_name": "NICD induces degradation of endogenous Snail protein",
"section_num": null
},
{
"section_content": "To understand the function of the NICD-Snail interaction, we infected primary MEFs with the MSCV driving Myc-NICD and/or Flag-Snail genes and evaluated Snail protein levels.Predictably, in Myc-NICD and Flag-Snail coinfected samples, NICD-induced Snail degradation was observed (Additional file 3).Given that Notch1 and Snail signaling individually regulate cell invasion, we considered whether their interaction might perturb their native functions [5,13].Using a cell invasion assay, we found that the number of invading Snail and NICD coinfected cells was significantly less than that of cells infected by Snail or NICD alone (Figure 6A).Parallel findings were observed in Huh7 and Hep3B cells in which coexpression of Snail and NICD suppressed invasiveness (Additional file 3 Figure 6B, and6C).Additionally, the number of invading cells was much greater in cells that had been transfected with Notch1 siRNA than those transfected with control siRNA (Figure 6D).However, the number of invading cells transfected with both Notch1 and Snail siRNAs did not increase (Figure 6D).To test whether NICD specifically inhibits Snail-induced cell invasion, we performed invasion assays in NICD and/or Snail siRNA-transfected Hep3B cells after treatment with H 2 O 2 .As portrayed in Figure 6E, we found that H 2 O 2 -treated cells ectopically expressing NICD were less invasive than H 2 O 2 -treated control cells, which correlates with the ability of NICD to inhibit H 2 O 2 -dependent upregulation of Snail expression (Figure 3E).However, in H 2 O 2 -treated cells cotransfected with Snail siRNA, NICD expression lacked any effect on cell invasion (Figure 6E and Additional file 3).Taken together, these results demonstrate that NICD can inhibit Snail-induced cell invasion. ",
"section_name": "Snail and NICD regulate the invasiveness of HCC cells",
"section_num": null
},
{
"section_content": "Previously, Notch1 was shown to cooperate with the Snail pathway by upregulating Snail transcription, inducing EMT and promoting hypoxia-induced tumor cell invasion [19].However, we found that NICD, which is an intracellular functional molecule liberated from Notch1, can suppress Snail protein via direct binding.This suggests that the Notch1-Snail pathway includes not only the single functional pathway but also a reciprocal interrelationship.Although both NICD and Snail protein play a central role in cancer cell growth, invasion and metastasis [5,[13][14][15], their physical interaction has not been described previously.Given the highly invasive phenotype of HCC and the overexpression of Snail in HCC tissue [7], we utilized HCC cell lines as a model cell system in which to study the functional involvement of NICD-Snail interaction in disease development. Notch1 is known to regulate Snail and Slug mRNA levels, but efforts have not been made to examine alternative functions of NICD and Snail expression in the same cancer cell line [19][20][21].In this study, we have demonstrated the ability of NICD to associate with Snail to induce its ubiquitination and proteasomal degradation.NICD has been reported to upregulate Snail activity by inducing Snail mRNA expression under hypoxic conditions [19].In addition, Notch1 is related to the mesenchymal program by activating Snail expression in cardiac development [22,23].Our findings appear to be inconsistent with these previous results.However, because Notch signals and cellular functions vary according to cell type and cellular environment, these inconsistencies could be caused by the different cell types and conditions.Although ectopic expression of NICD slightly induced Snail transcription, we postulate that the genetic interaction between Snail and NICD and their physical association may be physiologically exclusive events, which may account for their contrasting cellular effects.In our previous study, ROS stress upregulated Snail mRNA and protein expression [7].In the ROS-treated cells, Myc-NICD downregulated the Snail protein level, not the mRNA level (Figure 3).These latter data indicate that, under conditions of exogenous Snail expression or endogenous Snail upregulation by ROS stress, NICD reduces Snail protein levels by inducing Snail degradation without affecting Snail transcription.Because the NICD ΔANK mutant failed to induce ubiquitination and degradation of Snail, it appears that the physical interaction of NICD and Snail is required for the degenerative process.NICD is not known to have inherent E3 ligase functions, indicating that NICD may initiate serve as a cofactor of the degradation signal.We have shown that Myc-NICD interacts with MDM2 (Figure 4D), but the NICD ΔANK mutant did not (data not shown).In addition, in MDM2 siRNA-transfected cells, NICD-induced Snail degradation was decreased compared to control siRNA-transfected cells (Figure 4E), suggesting that MDM2 has a role in NICD-induced Snail degradation. We attempted to improve the transient transfection conditions with a stably NICD-expressing cell line based on a retroviral expression system.In these cells, the expression levels of NICD were much lower than those found with transient transfection.As shown in Figure 5 and Additional file 3 NICD-induced Snail degradation occurred in our stably NICD-expressing cells.In addition, to show that ligand-stimulated, NICD-induced Snail degradation took place, we used the cell line that expressed the exogenous Jagged1 ligand.In these cells, NICD induced Snail degradation (Figure 5C).Moreover, Notch1 siRNA upregulated endogenous Snail protein levels, as shown in Figure 5A.These data suggest that NICD-induced Snail degradation occurs in certain physiological conditions. In this study, NICD-induced degradation of Snail inhibited Snail-dependent invasive behavior, as expected.Consistent with these invasion data, E-cadherin expression decreased in Snail-overexpressed cells, but it did not in Snail-and NICD-coexpressed cells compared to control cells (Additional file 3).In our previous study, we reported p53-induced Snail degradation via MDM2 [18].Notch1 expression and signaling were regulated differently, depending on p53 status [13,24].In other systems, Notch1 related to the mesenchymal program by activating Snail expression [19,22,23].In another previous study, we showed that Notch1 and Snail differentially regulate invasion of HCC cells depending on p53 status [25].The inhibition of invasion by NICD and Snail coexpression was observed in both Hep3B cells (p53-null) and Huh7 cells (p53-mutant).On the other hand, in p53 WT cells such as HepG2, NICD and Snail coexpression promotes invasiveness [25].In the present study, we showed NICD regulated invasion via Snail degradation in p53-null or p53-mutant status.On the basis of these reports, we suspect that the interactions among p53, MDM2, Notch1 and Snail play an important role in EMT.Furthermore, these data explain the context-dependent regulation of EMT by the Notch signaling system.Our study provides one clue for understanding the complex regulation mechanism of p53, MDM2, Notch1 and Snail in the EMT process.The regulation of these proteins and their physiological contribution to HCC development and malignant behavior require further investigation.However, the mechanism that we have described presents substantial evidence of cross-interference between the Notch and Snail signaling pathways, which is mediated by their direct binding. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Herein we have identified NICD as a novel binding partner of Snail.Our results indicate that the interaction between NICD and Snail promotes Snail ubiquitination and degradation through an MDM2-dependent mechanism.Accordingly, we have shown that NICD impairs Snail-associated cell invasion in a conserved manner between MEFs and HCC cells.These findings collectively suggest that in the instance in which Notch1 and Snail are conjointly present, Notch signaling may serve as an antagonist of Snail function.The results of our research ultimately support caution in the use of genetic or pharmacological strategies that solely target the Notch pathway for therapeutic treatment in Snail-associated HCC.Further investigation is warranted to fully determine the precise cellular processes disrupted by Notch inhibition and thus to better assess the therapeutic value and clinical implications of Notch pathway antagonists. ",
"section_name": "Conclusions",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Hep3B human hepatoma cells, Huh7 human hepatoma cells, HT-29 human colorectal carcinoma cells, Panc-1 human pancreatic cancer cells, MDA-MB231 human breast carcinoma cells and 293T cells were cultured in DMEM with 10% fetal bovine serum (FBS).In some experiments, cells were incubated with 10 μM MG132 for 6 hours.To establish stable 293T lines expressing human triple-tagged Snail, we inserted the Snail cDNA sequence into the pIRES2-EGFP/SFB vector and transfected the 293T cells with either pIRES2-EGFP/SFB as a control or pIRES2-EGFP/SFB-Snail with FuGENE 6 transfection reagent (Roche Applied Science, Penzberg, Germany).G418 sulfate (Geneticin; Invitrogen, Carlsbad, CA, USA) or puromycin (InvivoGen, San Diego, CA, USA) were used to select colonies.Primary MEFs obtained from embryos of p53 -/-mice at day 13.5 were cultured in DMEM supplemented with 10% FBS.For siRNA experiments, cells were transfected with Notch1, Snail, MDM2 and control siRNA (ON-TARGETplus siRNA reagents; Dharmacon/Thermo Fisher Scientific, Lafayette, CO, USA) using Oligofectamine transfection reagent (Invitrogen/Life Technologies).After a 72-hour transfection or transduction, protein expression was analyzed by immunoblotting.All other reagents were purchased from Sigma-Aldrich (St Louis, MO, USA). ",
"section_name": "Cell culture and treatments",
"section_num": null
},
{
"section_content": "For purification, cells stably expressing Snail tagged with S protein-Flag-SBP (streptavidin-binding peptide) (SFB) were lysed with 50 mM Tris•HCl, pH 8.0, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA) and 0.5% Nonidet P-40 (NP-40) (NTEN) lysis buffer supplemented with protease and phosphatase inhibitors.Streptavidin Sepharose (first purification) followed by Sbeads (second purification) were used to bind SFBtagged Snail.Snail complexes were reduced, alkylated and digested overnight with trypsin.Peptides were characterized using MS/MS (Thermo Fisher Scientific, Inc, Waltham, MA, USA).The resulting peptide tandem mass spectra were searched against a comprehensive nonredundant protein database using SEQUEST Cluster software (Thermo Fisher Scientific, Inc). ",
"section_name": "Tandem affinity purification and mass spectrometry analysis",
"section_num": null
},
{
"section_content": "Human SNAI1 and NICD were obtained from the Huh7 cell line and cloned pCMV/HA, pCMV/Myc and/or p3 × Flag/CMV expression vectors.Using pCMV/HA-Snail as a template, a SNAG domain-deleted Snail construct (ΔSNAG), ZF region-deleted Snail construct (ΔZF) and ZF region construct were developed by performing PCR with the following primers: 5'-AGAATTCGGAAGCCCTCCGAC-3' (forward for ΔSNAG), 5'-AGCGGCCGCTCAGCGGGGACATCC-3' (reverse for ΔSNAG), 5'-AGAATTCCGCGCTC TTTCCTC-3' (forward for ΔZF), 5'-AGCGGCCGCT-CAT CGAGCCTGGAGATCCTTGGC-3' (reverse for ΔZF), 5'-AGAATTCCGAAGGCCTTCAACTGCAAA-TAC-3' (forward for ZF) and 5'-AGCGGCCGCT-CAGCGGGGACATCC-3' (reverse for ZF).Using pCMV/Myc-NICD as a template, RAM domain-deleted (ΔRAM), ANK domain-deleted (ΔANK), TAD domaindeleted (ΔTAD), OPA domain-deleted (ΔOPA) and PEST domain-deleted (ΔPEST) mutant constructs were developed by site-directed mutagenesis.All constructs were confirmed by sequencing.HA-tagged ubiquitin and human Jagged1 constructs were kindly provided by Dr C Chung and Dr L Nie, respectively.Flag-Snail WT and 2SA mutant constructs were obtained from Addgene, Inc (Cambridge, MA, USA). ",
"section_name": "Plasmids",
"section_num": null
},
{
"section_content": "Flag-Snail and Myc-NICD were cloned into pMSCV.To generate MSCV-expressing Flag-Snail or Myc-NICD, we transfected GP293 cells with pMSCV (for control virus), pMSCV/Flag-Snail or pMSCV/Myc-NICD with FuGENE 6 transfection reagent.Twenty-four hours after transfection the medium was changed, then the medium was collected at 12-hour intervals.The collected medium containing retrovirus was filtered and stored at -20°C.Before the retrovirus was used, we titrated it using the QIAamp Viral RNA Mini Kit (QIAGEN, Valencia, CA, USA) and the One Step SYBR PrimeScript RT-PCR Kit II (TaKaRa Bio Inc, Shiga, Japan).Cells were seeded at 30% confluence 12 hours before infection, and the media were replaced with medium containing MSCV.After infection for 24 hours, the medium was replaced with fresh medium and the infected cells were selected with puromycin (InvivoGen). ",
"section_name": "Retroviral expression vector and infection",
"section_num": null
},
{
"section_content": "In this study, we used the following antibodies: rat and mouse anti-Snail mAb (Cell Signaling Technology, Inc, Danvers, MA, USA), rabbit anti-Snail pAb (Abcam, Cambridge, MA, USA), rabbit anti-Notch1 mAb (Epitomics, Inc, Burlingame, CA, USA), goat anti-Notch1 pAb (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-Jagged1 pAb (Santa Cruz Biotechnology), rabbit anti-p21 pAb (Cell Signaling Technology, Inc, and Santa Cruz Biotechnology), mouse anti-E-cadherin mAb (BD Biosciences, Franklin Lakes, NJ, USA), mouse anti-Myc mAb (Santa Cruz Biotechnology), mouse anti-Flag mAb (Sigma-Aldrich), mouse anti-HA mAb (Sigma-Aldrich), rabbit anti-HA pAb (Abcam) and mouse anti-β-actin mAb (Sigma-Aldrich). ",
"section_name": "Antibodies",
"section_num": null
},
{
"section_content": "Tissue and cell lysates were prepared, and immunoblot analysis was performed as described previously [26].Band intensity was determined using ImageMaster 2D Elite version 4.01 software (Amersham/GE Healthcare, Uppsala, Sweden).For immunoprecipitation, Hep3B cells were transfected with Flag-Snail, Myc-NICD and MDM2.After 48 hours, the cells were lysed in buffer (50 mM Tris•HCl, pH 8.0, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA) and 0.5% Nonidet P-40 (NP-40)) and centrifuged at 16, 000 × g for 15 minutes to remove debris.Cleared lysates were subjected to immunoprecipitation with antibodies.For immunocytochemistry, cells were fixed in 4% paraformaldehyde at room temperature for 15 minutes, permeabilized in 5% Triton X-100 for 5 minutes, and then stained using pAbs.The secondary antibodies used were anti-mouse Alexa Fluor 594 dye conjugate and anti-rabbit Alexa Fluor 488 dye conjugate (Molecular Probes/Life Technologies, Carlsbad, CA, USA).Nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI blue) (Molecular Probes/Life Technologies).After mounting, the cells were visualized using a multiphoton confocal laser-scanning microscope (Carl Zeiss, Thornwood, NY, USA). ",
"section_name": "Immunoblot analysis, immunocytochemistry and immunoprecipitation",
"section_num": null
},
{
"section_content": "Twenty-four hours after transfection the cells were treated with 10 μM MG132 for 6 hours.The treated cells were then harvested with PBS containing 10 mM Nethylmaleimide (NEM) and 1 mM dithiothreitol (DTT).The cells were washed with PBS, centrifuged and subjected to one freeze-thaw cycle.Cell pellets were then resuspended in 200 μl of buffer 1 (10 mM Tris•HCl, pH 7.5, 10 mM NaCl, 0.5% NP-40, 5 mM EDTA, 1 mM ethylene glycol tetraacetic acid (EGTA), 10 mM NEM, 1 mM DTT, 5 mM NaF, 1 mM Na 3 VO 4 and protease inhibitor cocktail) and sonicated in a water bath (Bioruptor; Diagenode, Denville, NJ, USA).Next 500 μl of buffer 2 (20 mM Tris•HCl, pH 7.5, 0.5 M NaCl, 0.5% NP-40, 5 mM EDTA, 1 mM EGTA, 10 mM NEM, 1 mM DTT, 5 mM NaF, 1 mM Na 3 VO 4 and protease inhibitor cocktail) were added, and the extracts were subjected to a 30-minute rotation at 4°C.Extracts were then centrifuged.We added 2 μg of anti-Flag M2 antibody and protein A/G beads to the supernatant and experiments and edited manuscript.XQ out siRNA experiments.SMA carried out the interaction domain screen.HK participated in study design and gave critical discussions.DH edited manuscript.JKS provided mice for MEF, GJ designed study and edited manuscript.All authors read and approved the final manuscript. ",
"section_name": "In vivo ubiquitination assay",
"section_num": null
}
] |
[
{
"section_content": "This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (no.2010-0014611). ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "it for 2 hours.The beads were then washed three times, resuspended in loading buffer and boiled.Immunoblotting was performed as described above. Real-time PCR analysis of cDNA samples was performed with specific primers designed using Primer Express software (Applied Biosystems, Foster City, CA, USA).The primers used for Snail were 5'-AAGGATCTC-CAGGCT CGAAAG-3' (forward) and 5'-GCTTC GGATGTGCATCTTGA-3' (reverse) and the primers used for β-actin were 5'-GCAAAGACCTGTACGC-CAACA-3' (forward) and 5'-TGCATCCTGTCGG-CAATG-3' (reverse).Total RNA was extracted from cultured cells using an RNeasy kit (QIAGEN) according to the manufacturer's protocol.cDNA was synthesized using 1 μg of RNA with avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI, USA) and oligo(dT) primers.Transcript levels were assessed by quantitative real-time PCR (ABI 7300; Applied Biosystems), and all experiments were normalized to β-actin. Cell invasion was measured at 96 hours as described by the manufacturer (Oris Cell Invasion and Detection Assay Kit; Platypus Technologies, LLC, Madison, WI, USA).In the invasion assay, fluorescence was monitored at an excitation of 492 nm and an emission of 530 nm using a multiplate reader (EnVision Multilabel Reader; PerkinElmer, Inc, Waltham, MA, USA). The Duolink II fluorescence assay was used to analyze Hep3B, HT-29, Panc-1 and MDA-MB231 cells.Cells were seeded at 30% confluence on a cover glass and treated with 10 μM MG132 and 300 μM H 2 O 2 for 6 hours.Huh7 cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 for 15 minutes each, then blocked and incubated with mouse anti-Snail Ab (1:100 dilution; Cell Signaling Technology) and rabbit anti-Notch1 Ab (1:100 dilution; Epitomics, Inc) for 30 minutes.The Duolink II PLA probe protocol was used to detect the signals.After mounting, the cells were visualized using a multiphoton confocal laser-scanning microscope (Carl Zeiss). Bacterial lysate expressing GST-Snail proteins (WT, ΔZF and ZF mutants) was purified by immobilization on glutathione Sepharose beads (Pharmacia Biotech/GE Healthcare, Uppsala, Sweden).The beads were thoroughly washed with wash buffer (20 mM Tris•HCl, pH 8.0, 1 mM EDTA, 1 mM DTT, 150 mM NaCl, 1% Triton X-100) containing protease inhibitor mixture.The bound proteins were incubated with Myc-NICD-transfected cell lysate, washed with the wash buffer and eluted by boiling in the SDS sample buffer for 10 minutes.The sample was then analyzed by immunoblotting. The data in the bar graphs of Figure 3 and6 are expressed as the means (± SD) of three independent experiments.All results shown in the bar graphs are expressed as the fold ratio relative to untreated or control cells.Statistical analysis was performed using SPSS version 12 statistical software (SPSS Inc, Chicago, IL, USA). Additional file 1: Peptides sequence of identified Snail-bound proteins including Snail by mass spectrometry analysis.Abbreviations ANK: 6 ankyrin/cdc10 repeat; CSL: CBF1/RBPJk in vertebrates: Suppressor of hairless in Drosophila: Lag-1 in Caenorhabditis elegans; DMEM: Dulbecco's modified Eagle's medium; EMT: epithelial-to-mesenchymal transition; HCC: hepatocellular carcinoma; mAb: monoclonal antibody; MEF: mouse embryonic fibroblast; MS: mass spectrometry; MSCV: murine stem cell virus; MS/MS: tandem affinity purification and mass spectrometry; Mut: mutant; NICD: Notch1 intracellular domain; OPA: polyglutamine tract; pAb: polyclonal antibody; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PEST: proline-: glutamic acid-: serine-and threonine-rich region; ROS: reactive oxygen species; RT-PCR: reverse transcriptase polymerase chain reaction; siRNA: small interfering RNA; SNAG: divergent N-terminal region; TAD: transcriptional transactivation domain; WT: wild type; ZF: zinc finger. Author details 1 Department of Biological Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 151-747, Korea. 2 Department of Biological Science, Sungkyunkwan University, Seobu-ro, Jangan-gu, Suwon, Gyeonggi-do, 110-745, Korea. 3Laboratory of Developmental Biology and Genomics, College of Veterinary Medicine, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 151-747, Korea. Authors' contributions SOL carried out the majority of the experiments in this study, designed study, and wrote manuscript.HSK carried out protein interaction and siRNA The authors declare that they have no competing interests. ",
"section_name": "Real-time PCR analysis",
"section_num": null
},
{
"section_content": "it for 2 hours.The beads were then washed three times, resuspended in loading buffer and boiled.Immunoblotting was performed as described above. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Real-time PCR analysis of cDNA samples was performed with specific primers designed using Primer Express software (Applied Biosystems, Foster City, CA, USA).The primers used for Snail were 5'-AAGGATCTC-CAGGCT CGAAAG-3' (forward) and 5'-GCTTC GGATGTGCATCTTGA-3' (reverse) and the primers used for β-actin were 5'-GCAAAGACCTGTACGC-CAACA-3' (forward) and 5'-TGCATCCTGTCGG-CAATG-3' (reverse).Total RNA was extracted from cultured cells using an RNeasy kit (QIAGEN) according to the manufacturer's protocol.cDNA was synthesized using 1 μg of RNA with avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI, USA) and oligo(dT) primers.Transcript levels were assessed by quantitative real-time PCR (ABI 7300; Applied Biosystems), and all experiments were normalized to β-actin. ",
"section_name": "Real-time PCR analysis",
"section_num": null
},
{
"section_content": "Cell invasion was measured at 96 hours as described by the manufacturer (Oris Cell Invasion and Detection Assay Kit; Platypus Technologies, LLC, Madison, WI, USA).In the invasion assay, fluorescence was monitored at an excitation of 492 nm and an emission of 530 nm using a multiplate reader (EnVision Multilabel Reader; PerkinElmer, Inc, Waltham, MA, USA). ",
"section_name": "Invasion assays",
"section_num": null
},
{
"section_content": "The Duolink II fluorescence assay was used to analyze Hep3B, HT-29, Panc-1 and MDA-MB231 cells.Cells were seeded at 30% confluence on a cover glass and treated with 10 μM MG132 and 300 μM H 2 O 2 for 6 hours.Huh7 cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100 for 15 minutes each, then blocked and incubated with mouse anti-Snail Ab (1:100 dilution; Cell Signaling Technology) and rabbit anti-Notch1 Ab (1:100 dilution; Epitomics, Inc) for 30 minutes.The Duolink II PLA probe protocol was used to detect the signals.After mounting, the cells were visualized using a multiphoton confocal laser-scanning microscope (Carl Zeiss). ",
"section_name": "Duolink II fluorescence assay",
"section_num": null
},
{
"section_content": "Bacterial lysate expressing GST-Snail proteins (WT, ΔZF and ZF mutants) was purified by immobilization on glutathione Sepharose beads (Pharmacia Biotech/GE Healthcare, Uppsala, Sweden).The beads were thoroughly washed with wash buffer (20 mM Tris•HCl, pH 8.0, 1 mM EDTA, 1 mM DTT, 150 mM NaCl, 1% Triton X-100) containing protease inhibitor mixture.The bound proteins were incubated with Myc-NICD-transfected cell lysate, washed with the wash buffer and eluted by boiling in the SDS sample buffer for 10 minutes.The sample was then analyzed by immunoblotting. ",
"section_name": "Glutathione S-transferase pull-down assay",
"section_num": null
},
{
"section_content": "The data in the bar graphs of Figure 3 and6 are expressed as the means (± SD) of three independent experiments.All results shown in the bar graphs are expressed as the fold ratio relative to untreated or control cells.Statistical analysis was performed using SPSS version 12 statistical software (SPSS Inc, Chicago, IL, USA). ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "Additional file 1: Peptides sequence of identified Snail-bound proteins including Snail by mass spectrometry analysis.Abbreviations ANK: 6 ankyrin/cdc10 repeat; CSL: CBF1/RBPJk in vertebrates: Suppressor of hairless in Drosophila: Lag-1 in Caenorhabditis elegans; DMEM: Dulbecco's modified Eagle's medium; EMT: epithelial-to-mesenchymal transition; HCC: hepatocellular carcinoma; mAb: monoclonal antibody; MEF: mouse embryonic fibroblast; MS: mass spectrometry; MSCV: murine stem cell virus; MS/MS: tandem affinity purification and mass spectrometry; Mut: mutant; NICD: Notch1 intracellular domain; OPA: polyglutamine tract; pAb: polyclonal antibody; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PEST: proline-: glutamic acid-: serine-and threonine-rich region; ROS: reactive oxygen species; RT-PCR: reverse transcriptase polymerase chain reaction; siRNA: small interfering RNA; SNAG: divergent N-terminal region; TAD: transcriptional transactivation domain; WT: wild type; ZF: zinc finger. Author details 1 Department of Biological Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 151-747, Korea. 2 Department of Biological Science, Sungkyunkwan University, Seobu-ro, Jangan-gu, Suwon, Gyeonggi-do, 110-745, Korea. 3Laboratory of Developmental Biology and Genomics, College of Veterinary Medicine, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 151-747, Korea. Authors' contributions SOL carried out the majority of the experiments in this study, designed study, and wrote manuscript.HSK carried out protein interaction and siRNA ",
"section_name": "Additional material",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests. ",
"section_name": "Competing interests",
"section_num": null
}
] |
10.1038/s41598-017-01069-9
|
Paraquat poisoning induced pulmonary epithelial mesenchymal transition through Notch1 pathway
|
<jats:title>Abstract</jats:title><jats:p>Progressive pulmonary fibrosis is the most characteristic feature of subacute PQ poisoning. Epithelial-to-mesenchymal transition (EMT) is reported to be involved in the pulmonary fibrosis after PQ exposure. Recent evidence suggested Notch signaling is required for EMT. In this study, we investigated whether Notch1 and TGF-β1/Smad3 signaling was involved in EMT caused by PQ. It is demonstrated that A549 cells underwent EMT after treated with PQ at dose of 300 μmol/L for 6 days, charactered by increasing expression of mesenchymal marker α-SMA and decreasing expression of epithelial marker E-cadherin. We found that there was an apparent increased expression of Notch1 and jagged-1 in PQ induced EMT process. EMT could be enhanced by Jagged-1 ligand of Notch1, and be blocked by DAPT, a γ-secretase inhibitor. Our data also showed that the expression of TGF-β1/Smad3 increased after Notch1 is elevated in EMT caused by PQ. Jagged-1 significantly induced SMA expression, and this induction was completely inhibited by SB431542 in A549 cells. In conclusion, we demonstrated that Notch1 pathway was important in EMT induced by PQ, and TGF-β1/Smad3 signaling partly plays a role as the downstream of Notch1.</jats:p>
|
[
{
"section_content": "Paraquat (PQ, 1,1′-dimethyl-4,4′-bipyridinium), widely used as a herbicide in many developing countries around the world, can cause severe toxicity in human and animals.It is reported that PQ poisoning accounts for up to a third of all suicides worldwide 1 .After PQ poisoning accidentally or intentionally, patients develop acute multi-organ failure, followed by progressive pulmonary fibrosis and eventually death from respiratory failure.Progressive pulmonary fibrosis is the most characteristic feature of subacute PQ poisoning, which occurs over a period of days to weeks after PQ ingestion 2 .Although PQ-induced pulmonary fibrosis is associated with high mortality, the molecular mechanism of its toxicity is not yet established, and there are no effective antidotes available.It is widely understood that pulmonary fibrosis is a result of the proliferation of fibroblasts from profibroblasts and the subsequent accumulation of extracellular matrix proteins 3 .However, recent research has demonstrated that mesenchymal stem cells derived from the bone marrow 4,5 , and fully differentiated alveolar epithelial cells were capable of undergoing phenotypic switching to fibroblasts through a process termed the \"epithelial-to-mesenchymal transition\" (EMT) 6 . EMT is a process in which the polarized immotile epithelial cells are converted to motile mesenchymal cells through molecular reprogramming and phenotypic changes.Under physiological and pathological conditions, EMT is involved in the remodeling of tissues during embryonic development, tumor progression and development of fibrosis.During this transition, epithelial cells acquire the capacity to increase motility through down-regulating epithelial markers, such as E-cadherin, and up-regulating mesenchymal proteins, such as α-SMA [7][8][9] .Recent studies suggest the potential contributions of EMT in lung fibrosis [10][11][12] .Therefore, it is indeed possible that EMT may play an important role in pulmonary fibrosis caused by PQ poisoning.Recently, a study by Yamada et al. demonstrated the occurrence of EMT in lung fibrosis induced by PQ 13 . Previous reports indicated that Notch-mediated cell-to-cell signaling regulates development by controlling cell fate determination, cell proliferation, differentiation, and apoptosis during embryonic and postnatal stages 14,15 .Notch proteins are transmembrane receptors (Notch 1-4).Notch signaling is initiated by the interaction between the ligand (delta/jagged family) on one cell and the receptor on a neighboring cell.After ligand binding, γ-secretase-dependent proteolytic cleavage of the Notch receptor is triggered to release the Notch intracellular domain (NICD) to the nucleus which then activates target genes 16,17 . Recent evidence suggested that Notch signaling is involved in the EMT process.It has been reported that EMT is upregulated in fibrotic diseases of organs such as kidney, liver, and lungs in vivo [18][19][20] .Overexpression of NICD in podocytes can promote glomerular fibrosis in transgenic mice.Utilizing GSI, a γ-secretase inhibitor, relieved kidney fibrosis in mice 21 .In pulmonary fibrosis, activation of Notch pathway in alveolar cells could elevate expression of ACTA2 and COL1A1, and proliferation to myofibroblast 22 .Furthermore, blocking of Jagged1/Notch pathway by RNA interference, could effectively inhibit the switching from keratinocyte to mesenchymal cell 23 .Present investigations demonstrated that EMT induced by Notch signaling related to TGF-β, and TGF-β acts as the downstream of Notch signaling 24 . In the present study, we investigated whether Notch1 pathway is involved in EMT caused by PQ poisoning.Our data, for the first time, showed that Notch1 pathway was upregulated in EMT process after PQ poisoning in A549 cells in an in vitro cell culture model.Moreover, transforming growth factor-β (TGF-β1) was enhanced, and EMT was accelerated, after Jagged1 ligand overexpression in PQ poisoning.We also demonstrated that Smad-dependent signaling pathway was activated in response to PQ poisoning.These findings suggest that PQ poisoning active the Notch1 pathway by up-regulating the expression of TGF-β/Smad3, which resulted in the process of EMT. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "EMT in A549 cells, we exposed cells to different concentrations of PQ with different time in a preliminary experiment (not shown).We identified 300 μmol/L as the concentration at which EMT could occur with minimum toxicity to cells.So, in the subsequent experiments we used PQ at a concentration of 300 μmol/L.In the absence of PQ, A549 cells maintained epithelial morphology during the culture period.After exposure to PQ, the cells gradually displayed morphological appearances of mesenchymal cells.And after six days of PQ exposure, the majority of cells became spindle-shaped (Fig. 1A).These morphological features suggested that PQ may induce EMT-like phenotypic changes in A549 cells. During EMT process, epithelial cells acquire the capacity to increase motility through down-regulation of epithelial markers, such as E-cadherin, and up-regulation of mesenchymal proteins, such as α-SMA.The increase in α-SMA and decrease in E-cadherin expression is a marker of the occurrence of EMT.We found that after four days of PQ treatment, α-SMA mRNA levels were statistically increased in A549 cells as compared to day zero by qRT-PCR analysis.Meanwhile, E-cadherin mRNA levels were dramatically decreased after four days of PQ exposure (Fig. 1B). Western blot analysis revealed a similar decrease in E-cadherin and increase in α-SMA protein level in PQ-treated A549 cells, which correlated with mRNA changes.Specifically, PQ treatment for four days induced only a modest decrease in E-cadherin protein, while a dramatic decrease of E-cadherin protein was detected six days after PQ exposure (Fig. 1C). Decreased expression of E-cadherin and increased expression of α-SMA in PQ treated A549 cells were further confirmed by immunofluorescent staining.As shown in Fig. 1D, control A549 cells cultured in the absence of PQ predominantly expressed E-cadherin on the cell membrane, whereas cells in the presence of PQ gradually lost E-cadherin expression and increased expression of α-SMA. EMT induction in cells results in the acquisition of migration property.To evaluate the migration potential of A549 cells, a wound-healing assay was performed.The results showed a significantly increased migration capacity after PQ exposure by about 0.41 fold than that of normal A549 cells (Fig. 1E). All the data together revealed that A549 underwent EMT after PQ exposure at a dose of 300 μmol/L for six days. Notch1 signaling is required for PQ-induced EMT-like changes in A549 cells.Next, we investigated whether Notch signaling plays an important role in PQ induced EMT process.We examined mRNA of Notch1-4 and Jagged-1 ligand by qRT-PCR analysis after A549 cells were exposed to PQ for six days.The results showed that there was a significant increase in mRNA of Notch1, Notch3 and Jagged-1 (p < 0.05), while the mRNA level of Notch2 and Notch4 did not change apparently (Fig. 2A).Western blot analysis, which was carried out to detect the changes in expression of all the proteins above, showed that there was an apparent increase in expression of Notch1 and Jagged-1.Notably, Notch3 protein expression did not increase, which was not consistent with the qRT-PCR result.A similar result with Notch2 and Notch4 protein expression were observed in comparison to mRNA expression using qRT-PCR(Fig.2B).Then, we added Jagged-1 ligand at a dose of 500 ng/mL or DAPT, a γ-secretase inhibitor, at a dose of 10 μmol/L, to A549 cells exposed to 300 μmol/L PQ for six days.Then we collected the cells and detected the expression of EMT markers, α-SMA and E-cadherin, and Notch1 pathway related proteins. Upon stimulation with Jagged-1 ligand, we observed a significant increase in expression of α-SMA and Notch1, along with a decrease in E-cadherin protein by western blot analysis.However, after treated with DAPT, the relative amount of α-SMA and Notch1 decreased in comparison with PQ and PQ + Jagged-1 group (Fig. 2C).We also detected migration property of A549 cells in different conditions.A significantly increased migration capacity was observed after Jagged-1 stimulation, while a decreased capacity upon treated with DAPT (Fig. 3).The results revealed that Notch1 pathway is required in EMT induced by PQ poisoning, and it can be blocked by the γ-secretase inhibitor. PQ poisoning upregulates TGF-β/Smad3 pathway.TGF-β is implicated in the normal physiological repair as well as pathologic fibrotic processes of many organs, including the lungs 25,26 .TGF-β is also implicated in EMT during the fibrosis in several tissues, including lungs 27 .So, in the following step, we investigated the expression of TGF-β1 in EMT caused by PQ.We analyzed TGF-β1/Smad3 after a six day culture of A549 cells with PQ, PQ + Jagged-1, or PQ + DAPT.The expression level of TGF-β1/Smad3 was increased after PQ poisoning, and was enhanced by Jagged-1, while DAPT could inhibit the mRNA of TGF-β1/Smad3 as demonstrated by qRT-PCR(Fig.4A).Consistently, the result of western blot analysis indicated that Jagged-1 significantly increased the production of TGF-β1/Smad3, while DAPT decreased the expression of TGF-β1/Smad3 in A549 cells.In addition, the up-regulation of phosphorylated Smad3 protein was also induced by PQ alone and enhanced by Jagged-1 while blocked by DAPT in A549 cells (Fig. 4B). To determine whether Smad phosphorylation is required for EMT induced by PQ poisoning, we utilized 10 μmol/L of SB431542, a specific inhibitor of TGF-β receptor type I/ALK5 kinase that phosphorylates Smad2/3.A549 cells were cultured with PQ or PQ + Jagged-1, incubated in the presence or absence of SB431542 (10 μmol/L) for six days, and then harvested for western blot analysis.Western blot analysis showed that Jagged-1 significantly induced α-SMA, and this induction was completely inhibited by SB431542 in A549 cells (Fig. 4C).Consistently, fluorescence immunostaining showed that SB431542 effectively prevented the increase in SMA expression induced by Jagged-1(Fig.4D).Wound-healing assay also revealed apparently decreased migration behavior of A549 cells after treated with SB431542 (Fig. 3).Notably, expression of E-cadherin was not affected by SB431542.These results indicated that, in EMT caused by PQ, Notch1 acted through the induction of Smad3 phosphorylation. ",
"section_name": "PQ induced EMT-like phenotypic changes in A549 cells. To investigate whether PQ could induce",
"section_num": null
},
{
"section_content": "There was a significant increase in mRNA of Notch1, Notch3 and Jagged-1 after A549 cells were exposed to PQ for six days by qRT-PCR.(B) There was an apparent increase in expression of Notch1 and Jagged-1 protein by Western-blot analysis.(C) After treated with Jagged-1 ligand (ligand of Notch1, 500 ng/mL), a significant increase inα-SMA and Notch1, along with a decrease in E-cadherin protein by western analysis.However, after treating with DAPT (a γ-secretase inhibitor,10 μmol/L), α-SMA and Notch1 decreased than PQ and PQ + Jagged-1 group.*p < 0.05. ",
"section_name": "Figure 2. Notch1 signaling is required for PQ-induced EMT-like changes in A549 cells. (A)",
"section_num": null
},
{
"section_content": "PQ poisoning is a rising cause of death by insecticide poisoning, especially in developing countries.The mortality associated with PQ poisoning is increasing year by year.Most patients die from progressive pulmonary fibrosis following the acute phase of PQ poisoning.As there are no specific antidotes available as yet for PQ poisoning, in-depth investigation of the mechanism underlying progressive pulmonary fibrosis is utmost important.In this study, we exposed A549 cells to PQ and observed EMT-like phenotypic changes in A549 cells.Importantly, we demonstrate that Notch1 pathway is involved in EMT process after PQ poisoning, by up-regulating the expression of TGF-β/Smad3 signaling.This is the first investigation on the function of Notch pathway in EMT induced by PQ. It is reported that PQ induces EMT during pulmonary fibrosis in vitro and in vivo 13,28 .In our study, we also observed EMT-like phenotypic changes in A549 cells.After exposure to PQ at a dose of 300 μmol/L for six days, we observed spindle-shaped changes in the A549 cells.The expression of α-SMA, a marker of mesenchymal cells, increased, while, E-cadherin, a marker of epithelial cells, decreased, as observed by qRT-PCR, western blotting and immunofluorescent staining.Subsequently, we tested the migration capacity of A549 cells, which is an important mobility change associated with EMT.An increased migration capacity of about 0.41-fold than negative control cells was observed upon PQ exposure.All the data above provided vital clues to suggest that A549 cells were transforming and acquiring characteristics and behavior of mesenchymal cells in response to PQ exposure. Recent literature sheds light on the role of EMT, which occurs after PQ poisoning.However, the molecular mechanism of EMT induced by PQ poisoning is not yet established.To the best of our knowledge, our report is the first to demonstrate that Notch1 signaling is required for EMT induced by PQ poisoning.To explore the role of Notch1 in EMT process, we utilized Jagged-1 ligand to coactivate Notch1 signaling with PQ.Upon culture with Jagged-1, we observed changes in EMT-related markers such as α-SMA and E-cadherin which increased and decreased respectively.Meanwhile, when we used Notch1 signaling pathway inhibitor DAPT, the EMT process was blocked by the γ-secretase inhibitor.Migration capacity of A549 cells by wound healing assay was increased significantly after Jagged-1 stimulation, while decreased after DAPT treatment. Subsequently, we proceeded to detect TGF-β, the known strongest inducer of fibrosis, in EMT after PQ exposure.Our results indicating TGF-β1 in response to PQ exposure suggest that the activation of TGF-β1 should be involved in EMT response.Moreover, the activation of TGF-β1 by Jagged-1 and suppression by DAPT indicated the essential induction role of Notch1 on TGF-β1.Furthermore, in PQ induced EMT, by using SB431542, a specific inhibitor of TGF-β receptor type I/ALK5 kinase that phosphorylates Smad2/3, we demonstrate that α-SMA induction by Jagged-1 can be completely inhibited.We provide evidence that the Notch signal acts upstream from the TGF-β1/Smad3 signal in EMT induced by PQ. In our experiment, we demonstrated that Notch1/TGF-β1/Smad3 pathway is involved in the expression of α-SMA in EMT.However, the detailed functional point on α-SMA promoter zone is unknown, necessitating further advanced research on the α-SMA promoter. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "In conclusion, we demonstrated EMT-like phenotypic changes in A549 cells after PQ exposure.Notch1 signal was involved in the EMT process through TGF-β1/Smad3 signaling.This mechanism may contribute to the differentiation of myofibroblasts from alveolar epithelial cells during progressive pulmonary fibrosis caused by PQ poisoning. ",
"section_name": "Conclusions",
"section_num": null
},
{
"section_content": "Reagents.Paraquat dichloride hydrate, DAPT (N-(N-(3,5-difluorophenyl)-L-alanyl)-S-phenylglycine t-butylester) and SB431542 were purchased from Sigma-Aldrich (St. Louis, MO, USA).Recombinant human Jagged-1 Fc chimera protein was manufactured by R&D system (Minneapolis, MN, USA).Antibodies against NADPH, β-actin, TGF-β1 were purchased from Abcam (Cambridge, UK).Antibody against α-SMA and E-cadherin are provided by Santa Cruz Biotechnology (Dallas, TX, USA).Antibodies against Jagged-1, Notch1-4, Smad3 and phospho-Smad3 were purchased from Cell Signaling (Beverly, MA, USA).Cell culture and treatment.A549 human lung adenocarcinoma epithelial cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C in a humidified 5% CO 2 atmosphere. Before treatment, A549 cells were incubated in serum-free medium for 12 h.To evaluate the effects of Notch signal on EMT, 300 μmol/L PQ, 500 ng/mL recombinant Jagged-1 protein (a Notch1 protein ligand), and 10 μmmol/L DAPT (a γ-secretase inhibitor, 2 hour before stimulation by other reagents) were added to the cells.And 10 μmol/L SB431542, a specific inhibitor of TGF-β receptor type I/ALK5 kinase, were added to the cells at 2 hour before stimulation by other reagents.All the reagents need to be added again when changing medium. ",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Total cell RNA was isolated from A549 cells using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) and an ABI 7500 Fast real-time PCR instrument (Applied Biosystems, Warrington, UK) were used for quantitative real-time reverse transcription-PCR (qRT-PCR) with the gene-specific primer pairs listed in Table 1.The relative quantification results of gene expression were normalized to GAPDH transcript levels.For data analysis, the comparative threshold cycle (CT) value for GAPDH was used to normalize loading variations in the real-time PCRs. Western blot analysis.Following exposure to PQ, the cell lysates were prepared at the indicated time points using RIPA buffer.Protein concentration was determined by the Bradford assay.Equal amounts of proteins were transferred to a nitrocellulose membrane.After blocking, the membrane was incubated overnight at 4 °C with appropriate primary antibodies.Anti-E-cadherin, anti-α-SMA, anti-Notch1-4, anti-TGF-β1, anti-Smad3 and anti-phospho-Smad3 polyclonal antibodies were used at 1/1000 dilution.Then the membrane was washed with TBS-Tween and incubated with peroxidase-conjugated secondary antibodies at 1/4000 dilution.Immunoreactive proteins were visualized using enhanced chemiluminescence western blotting detection reagents and detected by HMIAS-2000 Imaging System.Band densities were determined by BioRad Quantity One software and quantified as the ratio of GAPDH protein expression. Monolayer wound-healing assay.After exposed to PQ for 6 days, A549 cells were collected for monolayer wound-healing assay.Briefly, A549 cells were grown in a 6-well plate, and linear wounds were made in the confluent monolayer using a 20 μL pipette tip.In each experiment, one well was used as a negative control with no treatment.The wounds were imaged 0 and 48 hours after wound creation.Corresponding wound areas were determined using Image-J and the remaining wound areas were calculated as a percentage of area at time 0 29,30 . Fluorescence immunostaining.A549 cells grown on cover slips were fixed with 4% paraformaldehyde for 10 min room temperature and stained with primary antibody: anti-E-cadherin or anti-α-SMA overnight at 4 °C, followed by the secondary antibody.Nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI).Fluorescence images were observed under a fluorescence microscope. Statistical Analysis.Data are presented as the mean ± standard deviation.The statistically significant difference was analyzed by independent sample t test for two groups, and ANOVA for three and more groups.SPSS16.0 software was used for statistical analysis.P < 0.05 was considered as statistically significant. ",
"section_name": "RNA Isolation and quantitative RT-PCR.",
"section_num": null
},
{
"section_content": "",
"section_name": "Gene",
"section_num": null
}
] |
[
{
"section_content": "This work was supported by the National natural science foundation of China (No. 81301627 and No. 81500628); and Liaoning Province Doctor Startup Foundation (No. 20131146).The authors express their appreciation to Dr. Mingkai Xu for helpful advice on migration assays. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "T.L. designed the experiment and prepared figure 1. X.Y., S.X. and Y.C. prepared figure 23.N.W. designed the experiment, gathered data and wrote the main manuscript text.All authors reviewed the manuscript. Competing Interests: The authors declare that they have no competing interests. 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": "T.L. designed the experiment and prepared figure 1. X.Y., S.X. and Y.C. prepared figure 23.N.W. designed the experiment, gathered data and wrote the main manuscript text.All authors reviewed the manuscript. ",
"section_name": "Author Contributions",
"section_num": null
},
{
"section_content": "Competing Interests: The authors declare that they have no competing interests. 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.13712
|
Humoral immune responses toward tumor-derived antigens in previously untreated patients with chronic lymphocytic leukemia
|
In chronic lymphocytic leukemia (CLL) the occurrence and the impact of antibody responses toward tumor-derived antigens are largely unexplored. Our serological proteomic data show that antibodies toward 47 identified antigens are detectable in 29 out of 35 patients (83%) with untreated CLL. The glycolytic enzyme alpha-enolase (ENO1) is the most frequently recognized antigen (i.e. 54% of CLL sera). We show that ENO1 is upregulated in the proliferating B-cell fraction of CLL lymph nodes. In CLL cells of the peripheral blood, ENO1 is exclusively expressed at the intracellular level, whereas it is exposed on the surface of apoptotic leukemic cells.From the clinical standpoint, patients with progressive CLL show a higher number of antigen recognitions compared to patients with stable disease. Consistently, the anti-ENO1 antibodies are prevalent in sera from patients with progressive disease and their presence is predictive of a shorter time to first treatment. This clinical inefficacy associates with the inability of patients' sera to trigger complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity against leukemic cells.Together, these results indicate that antibody responses toward tumor-derived antigens are frequently detectable in sera from patients with CLL, but they are expression of a disrupted immune system and unable to hamper disease progression.
|
[
{
"section_content": "Immune dysfunctions are a key feature of chronic lymphocytic leukemia (CLL) and frequently result in clinically manifest complications contributing to patients' morbidity and mortality, such as opportunistic infections and autoimmune conditions.The impairment of the host immune system also correlates with disease progression and may reflect the attempt of the leukemic cells to evade immune control [1]. Perturbations of both cellular and humoral immunity are observed in CLL patients.The T-cell compartment is highly abnormal, with changes in the phenotype and absolute number of circulating CD4+ and CD8+ T cells and with the accumulation of terminally differentiated effector memory T cells [2].T lymphocytes express Research Paper markers of exhaustion, such as upregulated PD-1, leading to a pronounced Th2 skewing of the T-cell responses [3], have reduced cytotoxic functions and an impaired formation of immune synapses with antigen (Ag) presenting cells [4].Regulatory T cells are increased in the peripheral blood (PB) of patients with advanced disease [5], and Vγ9Vδ2 T cells display features of exhaustion and are negative prognosticator of a shorter time to first treatment (TTFT) [6]. Quantitative and qualitative changes of the humoral responses are also very common in patients with CLL.Hypogammaglobulinemia affects up to 85% of patients and its severity correlates with stage and duration of the disease and with the susceptibility to recurrent infections [1,7].On the other hand, haematopoieticspecific auto-antibodies (Ab) are frequently observed and autoimmune haemolitic anemia (AIHA) and/or immune thrombocytopenia are rather frequent complications.Autoimmunity is usually due to high affinity polyclonal immunoglobulin (Ig) G auto-Ab that are produced by the non-malignant B lymphocytes and recognize red cells-or platelets-derived auto-Ag [4,8]. So far, little is known on the occurrence of humoral immune responses toward tumor-derived Ag in CLL.In a previous report, Krackhardt et al. detected circulating Ab recognizing 14 Ag derived from primary tumor samples in sera from patients with CLL [9].However, the biologic functions and the clinical impact of spontaneously occurring humoral responses directed toward tumorderived Ag have not been investigated. Serological Proteome Analysis (SERPA) is a valuable method to detect Ag-specific Ab responses in human malignancies.SERPA combines electrophoretic separation of proteins from tumor cells, western blotting and mass spectrometric (MS) identification of Ag recognized by sera.SERPA allowed the identification of tumor-derived Ag able to trigger humoral immune responses in several solid [10][11][12][13][14][15][16] and hematologic tumors [17][18][19][20].Dubovsky et al. have recently applied a modified SERPA to CLL to define new membrane-associated targets, and identified lymphocyte cytosolic protein 1 as an important factor in chemokine-induced migration of leukemic cells [21]. The aim of our study was to investigate the occurrence of humoral responses toward Ag derived from primary tumor cells in sera from previously untreated patients with CLL, also evaluating their cytotoxic function and the correlation with parameters of disease evolution. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "Sera obtained from 35 patients with previously untreated CLL were individually screened for the presence of IgG-based immunoreactivity against proteins derived from autologous CLL cells (Figure 1A and1B).To verify the CLL-specificity of the detection, 8 CLL proteomic maps (patients 05, 06, 10,11,12,13,16,29) were individually probed with a different healthy donor's (HD) serum (data not shown).Overall, the 35 CLL sera recognized 144 Ag (mean:4.1, min:0, max:20), whereas the 8 HD sera recognized only 3 Ag (mean:0.4,min:0, max:1) (p=0.007). The MS analyses of the 144 Ag spots produced by CLL sera led to the identification of 47 proteins that were recurrently recognized by patients' sera.Table 1 summarizes the chemical features, spectrometric data, and frequency of recognition of 18 Ag, which Sera from progressive CLL are more immunoreactive than sera from stable CLL The immunoreactive status of patients was not associated with gender, age, Rai or Binet stage, lymphocytosis, monocyte and platelet counts, hemoglobin, β2-microglobulin or lactate dehydrogenase values, percentage of CD38+ or ZAP70+ cells, immunoglobulin heavy chain variable regions (IGHV)mutational status, fluorescence in situ hybridization data, hypogammaglobulinemia, autoimmunity, concurrent infections or allergies.Our cohort included 10 patients in active disease progression and 22 patients with a stable disease.For the remaining 3 patients, the disease status at the time of SERPA was not available.Interestingly, we found that patients with progressive CLL showed a significantly higher number of Ag recognitions compared to patients with stable disease (p=0.01)(Figure 2).Overall, median TTFT of patients was 29 months and median overall survival (OS) was not reached at the median follow-up of 81 months.The immunoreactivity status was not a statistically significant TTFT or OS predictor. ",
"section_name": "Immunoreactivity of CLL sera toward CLLderived Ag and protein identification",
"section_num": null
},
{
"section_content": "To determine whether the circulating Ab detected by SERPA in patients' sera were partly produced by a class-switched sub-clone derived from the leukemic clone, we first analyzed the IGHV repertoire of patients who shared the same Ag recognitions.Patients with similar immunoreactivities did not exhibit the same IGHV rearrangements.In addition, the analysis of the complementarity determining region 3 (CDR3) revealed that 6 out of 35 CLL patients displayed stereotyped B-cell receptor (BCR) and belonged to 4 subsets, with no association between stereotypy and Ag recognition.As a confirmatory evidence, we cloned and produced a soluble derivative of the leukemic BCR (ScFv-Fc) from patient 10 and blotted it in parallel with the whole patient's serum on two identical autologous proteomic maps.The patient's serum recognized 5 Ag, but none of these was also recognized by the autologous tumor-derived ScFv-Fc (Supplementary Figure S1).Taken together, these results indicate that the Ab detected by SERPA are entirely produced by the normal B-cell population, and do not include a soluble fraction of the leukemic BCR. ",
"section_name": "Circulating Ab are produced by the polyclonal B-cell population and not by the leukemic clone",
"section_num": null
},
{
"section_content": "ENO1 was the most relevant Ag recognized by patients' sera, since ENO1-specific circulating Ab were detectable in 19 out of 35 (54%) CLL sera and in none of the HD sera (p=0.006)(Table 1).To confirm the MS identification of the protein, we incubated the proteomic maps obtained from the tumor cells of 10 anti-ENO1 Ab+ (patients 22,23,24,25,26,27,29,31,32,33) and 4 anti-ENO1 Ab-patients (patients 28, 30, 34, 35) with a commercially available anti-ENO1 polyclonal Ab.The anti-ENO1 Ab generated the same Ag spots produced by the sera of patients with CLL (Supplementary Figure S2). ENO1 expression pattern was investigated by immunohistochemistry and multicolor immunofluorescence confocal microscopy in lymph node (LN) sections obtained from 3 CLL and 3 reactive (R) LN.The CLL LN displayed an almost complete effacement of the normal architecture by CLL cells and evidence of morphologically distinct pseudo-follicles, comprising areas rich in prolymphocytes and paraimmunoblasts.The anti-ENO1 Ab stained all CLL and R LN, and higher magnification indicated an increased expression in correspondence of proliferating cells of both CLL and R LN sections, at least on the basis of cell morphology (Figure 3A and3B).Multicolor tissue immunofluorescence was then used to determine which cells were mostly ENO1+.The anti-ENO1 Ab was combined with anti-CD2 and anti-CD23 Ab to detect T and B cells, respectively.In the CLL LN ENO1 reactivity was mostly associated to the CD23+ population, while in the R LN it was mostly associated to the CD2+ T cells (Figure 3C-3E).Ki67 was then used to identify proliferating cells in both tissues.Within the CLL LN a significant proportion of proliferating B cells was apparent and these cells were intensely ENO1+.Conversely, in the reactive LN the most of the proliferating cells were CD2+, and they also showed ENO1 reactivity, suggesting that ENO1 is highly expressed by proliferating cells, independently of the lineage (Figure 3D-3K). ",
"section_name": "Alpha-enolase (ENO1) is the most frequently recognized Ag and is overexpressed by proliferating CLL cells of the LN",
"section_num": null
},
{
"section_content": "ENO1 expression was also evaluated in the PB compartment.First, we compared the intracellular expression of ENO1 in PB mononuclear cells (PBMC) subpopulations from CLL patients.Cytofluorimetric analysis showed that CD19+/CD5+ CLL cells, CD14+ 4A).We then evaluated the membrane expression of ENO1.ENO1 was not expressed on the surface of CD19+/ CD5+ leukemic cells, whereas it was expressed to higher extent by CD14+ monocytes, and in a proportion of CD3+ T lymphocytes (Figure 4B).After 4 days of in vitro culture, the mean fold increase in the percentage of cells expressing ENO1 on the surface was significantly higher in the apoptotic (Annexin-V+ [AnnV+]) than in the viable (AnnV-) fraction of CD19+ cells (p=0.02)(Figure 4C).This difference in ENO1 surface expression (mean fold increase) was not observed between the apoptotic and viable fraction of CD19-cells (Figure 4D). ",
"section_name": "ENO1 is transferred on the surface of CLL cells undergoing apoptosis",
"section_num": null
},
{
"section_content": "The presence of circulating anti-ENO1 Ab was not associated with the occurrence of clinically evident autoimmune manifestations.By contrast, ENO1 reactivity was significantly associated to parameters of disease progression such as higher lymphocyte counts (p=0.02) and lower platelet numbers (p=0.007)(Supplementary Figure S3A andS3B).Similarly, anti-ENO1 Ab (Figure 5A) were more frequently detected in sera from patients with progressive disease than in sera from patients with stable disease (p<0.0001).As expected, log-rank test indicated that the presence of anti-ENO1 Ab in patients' sera was a negative TTFT predictor in univariate analysis: the median TTFT of anti-ENO1 Ab+ CLL patients was 7 months, whereas that of anti-ENO1 Ab-CLL patients was 107 months (p=0.01)(Figure 5B).Lastly, we observed that the total level of serum IgG was significantly lower in anti-ENO1 Ab+ compared to anti-ENO1 Ab-CLL sera (p=0.02)(Supplementary Figure S4). ",
"section_name": "Immunoreactivity toward ENO1 associates with CLL progression and shorter TTFT",
"section_num": null
},
{
"section_content": "Next, we asked whether the circulating Ab identified in patients sera were effective in triggering CDC.We first tested the efficacy of our samples of CLL sera as source of complement.As expected, the viability of CLL cells exposed to the monoclonal Ab alemtuzumab was significantly reduced compared to untreated controls (p<0.01).By contrast, we did not observe any significant decrease in cell viability when CLL cells were exposed to patients' sera, in the absence of alemtuzumab (Figure 6A-6D).Flow cytometry revealed that there was no deposition of the complement component 4 on the surface of CLL cells after incubation with patients' sera (data not shown).Overall, these data show that the circulating Ab detected by SERPA in CLL sera are not able to induce CDC toward CLL cells. We then performed CDC assays with sera from anti-ENO1 Ab+ patients and the U937 cell line, which expresses high levels of surface ENO1 (Figure 6E).Again, no modification of cell viability was observed when U937 cells were treated with anti-ENO1 Ab+ sera, even when sera were used at 100% concentration (Figure 6F and6G).These data demonstrate that the circulating Ab do not trigger CDC at the in vivo concentrations, even when the (I,J).Cumulative analysis of ENO1 mean pixel intensity (arbitrary units) confirmed that the CD3+/Ki67+ proliferating fraction of the T-cell compartment expressed significantly higher levels of ENO1 than CD3+/Ki67-resting fraction, in both CLL and R LN (p<0.001 and 0.03 respectively) (K).For cumulative data, at least 3 randomly chosen fields from 3 different samples were analyzed.Samples were analyzed using a TCS SP5 laser scanning confocal microscope (Leica Microsystems) with an oil immersion 63x/1.5 objective lens, images were acquired with LAS AF Version Lite 2.4 software and processed with Photoshop (Adobe Systems).ENO1 mean pixel intensity was calculated with ImageJ software (http://rsbweb.nih.gov/ij/).Box and whiskers plots represent median values, first and third quartiles, and minimum and maximum values for each dataset.Statistical analysis was performed using Mann-Whitney U test.ENO1 was not expressed on the surface of CD19+/CD5+ CLL cells, whereas it was widely expressed by CD14+ monocytes and in a proportion of CD3+ lymphocytes.C, D. Surface ENO1 fold increase expression in CD19+ and CD19-cell fractions.Fold increase was calculated for each patient as a ratio between the percentage of CD19+/ENO1+ detected after 4-day in vitro culture and the percentage of CD19+/ENO1+ cells at baseline.After 4 days of culture, the fold increase in ENO1 expression was significantly higher in the apoptotic (AnnV+) than in the viable (AnnV-) fraction of CD19+ cells (p=0.02)(C).By contrast, there was no difference in the fold increase of ENO1 expression between the apoptotic (AnnV+) and the viable (AnnV-) fraction of CD19-cells (D).Box and whiskers plots represent median values, first and third quartiles, and minimum and maximum values for each dataset.cognate Ag is highly expressed on the surface of the target cells. In the last set of experiments, we tested the ability of anti-ENO1 Ab+ sera to trigger ADCC toward both the U937 cell line and primary CLL cells.We found that the % of ADCC of U937 cells cultured for 18 hours in the presence of 10% diluted anti-ENO1 Ab+ serum and HD PBMC, used as effector cells, was not significantly increased compared to target cells cultured with PBMC alone, in the absence of serum.Similar results were obtained using CLL cells as targets (Figure 7A-7D).Therefore, we can conclude that the circulating Ab detected by SERPA are unable to trigger ADCC against leukemic cells. ",
"section_name": "Serum Ab are not able to trigger complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC)",
"section_num": null
},
{
"section_content": "In CLL, cellular immune responses are unable to respond to the tumor and they can even support disease evolution [22].The impact of humoral responses toward tumor-derived Ag in CLL is largely unexplored.Through the use of SERPA we show that 83% of CLL sera have circulating Ab toward at least one CLL-derived Ag.Interestingly, we observe that sera from progressive CLL are significantly more immunoreactive than sera from stable CLL. The circulating Ab detected by SERPA are expression of an immune response generated by the residual polyclonal B-cell population toward self-Ag.We do not observe shared Ag recognition between a representative CLL serum and the ScFv-Fc derived from the autologous leukemic BCR.On the other hand, patients who share the same Ag recognitions do not show recurrent IGHV rearrangements or BCR stereotypies.Autoimmune phenomena are rather frequent complications of CLL and mostly result in autoimmune cytopenias [23].A clear association between autoimmune cytopenias and poor prognostic variables, such as high β2-microglobulin, CD38 and ZAP70 expression, and unmutated IGHV status, has already been described [24][25][26].Moreover, direct antiglobulin test positivity, even in the absence of overt AIHA, associates with adverse prognostic factors and shorter OS [27].In our cohort, we observe a significantly higher degree of immunoreactivity in patients with progressive CLL than in patients with stable disease, even though the immunoreactive status is not related to adverse prognostic factors. MS analyses show that the Ab detected by SERPA mostly recognize intracellular Ag, such as glycolytic enzymes, cytoskeletal elements and ribonucleoproteins.Among others, ENO1, a glycolytic enzyme, is the most relevant Ag, detected in 19 out of 35 (54%) CLL sera.The overexpression of ENO1 is associated with tumor development through a process known as Warburg effect [28].The Warburg effect is a metabolic reprogramming, which consists in a positive regulation exerted by the hypoxia-inducible factor (HIF) on the expression of glycolytic enzymes, such as ENO1, occurring when cancer cells are exposed to hypoxic conditions [29,30].Interestingly, CLL cells express the oxygen-regulated HIF-1α subunit even under normoxia [31], and the expression and the transcriptional activity of HIF-1α are further upregulated by exposure of CLL cells to stromal cells [32].In the LN, proliferating CLL cells are confined to specialized structures called pseudo-follicles, where they interact with T lymphocytes and stromal cells.Our data show that ENO1 is more expressed in the leukemic cells of the CLL LN than in the normal B cells of the R LN, and confirm that it is more expressed by the proliferating B cells of the pseudo-follicles than by the resting B-cell fraction. Due to its tumor-related overexpression and ability to induce humoral and/or cellular immune responses, ENO1 has already been classified as a tumor-associated Ag in solid cancers, such as pancreatic ductal adenocarcinoma (PDA) [12,33].Cappello et al. have reported that a DNA vaccine coding for ENO1 can elicit anti-ENO1 IgG Ab which are able to induce the killing of murine PDA cells by CDC [33].Unexpectedly, in our cohort of CLL patients, the presence of anti-ENO1 Ab is prevalent in sera from patients with progressive CLL and is predictive of a shorter TTFT.One possible reason of the different clinical impact exerted by anti-ENO1 Ab in solid tumors and in CLL is that ENO1 is highly expressed at the intracellular level, but is not expressed on the cell membrane.It is not clear how intracellular self-Ag can become immunogenic and trigger humoral responses.One possible explanation is that overexpressed self-proteins are exposed during the apoptotic turnover of the leukemic cells, thus becoming visible and capable of inducing autoreactive immune responses.This hypothesis is corroborated by our data showing that after 4 days of in vitro culture, the mean fold increase in the percentage of cells expressing ENO1 on the surface is significantly higher in the apoptotic than in the viable fraction of CD19+ cells.The higher frequency of immunoreactivities detected by SERPA in the sera of patients with progressive CLL can be explained by the observation that in advanced-stage CLL the malignant clone, which in early-stage is arrested in G0/G1, evolve into an autonomously proliferating cell population showing a greater ability to enter spontaneous apoptosis [34].In the appropriate context, such as in the spleen and LN, where T cells may induce the activation and Ag presentation by B cells, this turnover potentially leads to a greater exposition of intracellular overexpressed Ag and to the production of autoreactive Ab. To functionally characterize the humoral responses detected by SERPA we evaluated the in vitro ability of patients' sera to kill primary CLL cells by CDC or ADCC.Deficiencies or reduced levels in one or more complement components have already been reported in CLL patients [35,36].However, we demonstrated that CLL sera behave as proper source of complement, in inducing alemtuzumab-mediated CDC as effectively as HD sera.By contrast, in the absence of alemtuzumab, polyreactive CLL sera are not able to induce complement deposition on the surface of CLL cells and do not trigger CDC toward primary tumor cells.Similarly, CLL sera do not trigger ADCC against CLL cells, in the presence of PBMC isolated from HD used as effector cells.A further demonstration of the functional incompetence of circulating Ab detected by SERPA is the observation that CLL patients with immunoreacitive sera, even those with polyreactive sera, lack clinically manifest autoimmune phenomena.A number of reasons may underlie this functional incompetence.First of all, the tumor Ag recognized by the circulating Ab are mainly intracellular proteins and therefore are not optimal targets for an Abmediated immune response.However, we do not observe any cytotoxicity even when the U937 cell line, which expresses high levels of surface ENO1, is used as target for the CDC and ADCC assays.In line with these results is the observation that the absolute number of monocytes, which also express high levels of surface ENO1, is not decreased in the PB of CLL patients with anti-ENO1 circulating Ab (data not shown).These findings show that Ab-related issues, such as low sera concentrations of the circulating Ab and/or low binding affinity toward the corresponding Ag, may also contribute to the functional incompetence of the humoral responses detected by SERPA. Overall, our results show that the Ab responses detected by SERPA are an epiphenomenon of a disrupted immune system, which is unable to control disease evolution.It has already been reported that the chronic inflammation state determined by an enhanced and persistent activation of humoral immune responses, in combination with the suppressed cellular anti-tumor immunity, may favour the tumor progression and support disease evolution [37].In this context, it will be of interest to determine the immunomodulatory properties of the new BCR inhibitors, which have now been introduced in the clinical practice for the treatment of CLL.Current studies already show a recovery of humoral immunity and normal B-cell numbers in patients on ibrutinib, leading to a decrease in the rate of infections [38].This observation, together with the promising results obtained in experimental models of autoimmune diseases [39,40], support the hypothesis of an ibrutinib-induced immunomodulation which may contribute to tumor control. ",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "PB samples were collected from a total of 86 patients with untreated CLL, after their informed consent, in accordance with the Declaration of Helsinki and approval by the local institutional review board.Sections of LN infiltrated by CLL cells and reactive R LN were obtained from the Department of Medical Sciences of the University of Torino, Italy.The diagnosis and progression of CLL were defined according to International Workshop on CLL-National Cancer Institute (IWCLL/NCI-WG) guidelines [41].The disease was defined stable in absence of signs of progression.A cohort of 35 patients with CLL was analyzed by SERPA.Patients' biological and clinical features were collected, when available, by chart review (Supplementary Table S2).The median follow-up of all patients was 81 months.The IGVH mutational status and the CDR3 clustering analyses were performed as previously reported [42,43].The control group consisted of 12 HD kindly provided by the local blood bank. ",
"section_name": "Patient population",
"section_num": null
},
{
"section_content": "PBMC and purified B lymphocytes were prepared as described [32].Serum samples were isolated from PB by centrifugation and stored at -80°C until use.In selected experiments, the human cell line U937 (ATCC, CRL 1593.2) was used. ",
"section_name": "Cells and serum samples",
"section_num": null
},
{
"section_content": "Pellets obtained from at least 10 7 purified CLL cells were solubilized in lysis buffer (urea 9M, CHAPS 4%, Na 3 VO 4 1mM, DTT 80mM, protease inhibitors and nuclease).Samples were spun down at 13800 g for 10 minutes at 4°C.The clear supernatant was quantified with DC Protein assay kit (Bio-Rad, Hercules, CA, USA).2-DE on ready-made IPG strip (7-cm IPG strips, pH 3-10NL; Bio-Rad) was performed essentially as described [44].The 2-DE maps were obtained in duplicate and stained with Coomassie Blue or transferred onto a nitrocellulose membrane (GE Healthcare Biosciences GmbH, Uppsala, Sweden) for serological analysis.The 2-D gel images were acquired using the ChemiDoc MP system (Bio-Rad). ",
"section_name": "Sample preparation and 2-Dimension Electrophoresis (2-DE)",
"section_num": null
},
{
"section_content": "The membrane was incubated overnight at 4°C with serum, as primary Ab at working dilution of 1:100, with a single chain variable fragment-fragment crystallisable region (ScFv-Fc) or with an anti-ENO1 Ab (Santa Cruz Biotechnology, Inc; CA, USA).Then the membrane was incubated with an anti-human (Santa Cruz Biotechnology, Inc) or anti-mouse (GE Healthcare Biosciences GmbH) IgG HRP-conjugated Abs.Images were acquired by the ChemiDoc MP system (Bio-Rad). ",
"section_name": "Western blot analysis",
"section_num": null
},
{
"section_content": "MS analysis was performed as described [44].The 25 most intense masses were used for database searches against the Swiss-Prot database using the free search program Mascot (http://www.matrixscience.com).Only proteins with a Mascot score greater than 56 were taken into consideration. ",
"section_name": "Protein identification by MS",
"section_num": null
},
{
"section_content": "VH and VL genes were amplified with specific primers from CLL derived cDNA as already described [45].V genes were assembled as ScFv and cloned in fusion with the human Fc region (as scFv-Fc) in the pUCOE vector [46].CHO-S cell line was transfected and stable clones was obtained for ScFv-Fc expression.Ab produced in cell culture supernatants were purified using a protein A/G column. ",
"section_name": "ScFv-Fc production",
"section_num": null
},
{
"section_content": "ENO1 surface and intracytoplasmic expression was evaluated cytofluorimetrically on B cells, T cells and monocytes in PB samples as reported in Supplemental Methods. ",
"section_name": "Flow cytometry",
"section_num": null
},
{
"section_content": "PBMC from patients with CLL were cultured as described [32].The percentage of viable and apoptotic cells was determined by Annexin-V (AnnV) or AnnV/ Propidium Iodide (PI) staining with the MEBCYTO-Apoptosis Kit (MBL Medical and Biological Laboratories, Naka-ku Nagoya, Japan). ",
"section_name": "Cell culture and viability assay",
"section_num": null
},
{
"section_content": "Cell morphology and numbers were studied by Giemsa staining.For immunocytochemistry, coverslips were stained as detailed in Supplemental Methods.Formalin-fixed, paraffin-embedded sections of CLL or R LN were stained and analyzed by light microscopy as already described [47] (details in Supplemental Methods). ",
"section_name": "Immunohistochemistry and confocal immunofluorescent microscopy",
"section_num": null
},
{
"section_content": "For CDC, purified CLL cells (2 x 10 5 ) were incubated in presence or absence of alemtuzumab (10 μg/ml) for 30 minutes at room temperature.CLL cells were then washed with PBS and incubated with selected patients' sera at different dilutions for 60 minutes at 37°C.U937 cells were incubated only with CLL patients' sera.Cell viability was evaluated using AnnV/PI staining (details in Supplemental Methods).For ADCC, purified CLL cells or U937 cells, used as target cells (T), were labeled with CFSE (5 μM) for 30 minutes at 37 °C.CLL cells and U937 cells were then incubated with PBMC from healthy donors, used as effector cells (E) for 18 hours at 30:1 E:T ratio, in the presence or absence of 10% diluted CLL patients' sera.At the end of the co-culture target cells viability was evaluated by PI staining.% of ADCC was calculated as follow: % PI (T + E + serum) -% PI (T) / % PI (triton X treated T) -% PI (T). ",
"section_name": "CDC and ADCC assays",
"section_num": null
},
{
"section_content": "Statistical analyses were performed with GraphPad Prism (San Diego, CA, USA).Continuous variables were compared by Mann-Whitney U (unpaired data) or Wilcoxon signed rank (paired data) tests.The χ 2 test was used in case of dichotomous variables.OS and TTFT were defined as the time between the date of SERPA and, respectively, the date of death or last follow-up and the date of first-line treatment or the last follow-up.OS and TTFT were estimated by the Kaplan-Meier method and the difference between groups was assessed by log-rank test.A p value <.05 was considered significant. ",
"section_name": "Statistical analysis",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "CONFLICTS OF INTEREST",
"section_num": null
},
{
"section_content": "",
"section_name": "Authorship contributions",
"section_num": null
}
] |
10.21203/rs.3.rs-1305178/v1
|
Clinical Significance of Notch Receptors In Triple Negative Breast Cancer
|
<jats:title>Abstract</jats:title> <jats:p><jats:bold>Background: </jats:bold>The Notch signaling pathway is an evolutionary conserved cell signaling pathway that plays an indispensable role in essential developmental processes. Aberrant activation of Notch pathway is known to initiate wide array of diseases and cancers. The role of Notch receptors and the effect of their expression in Triple Negative Breast Cancer is unknown. <jats:bold>Methods and Results:</jats:bold> In this study, we evaluated the association between Notch receptors and clinicopathological parameters including disease-free survival and overall survival of one hundred TNBC patients by immunohistochemistry. We observed membrane, cytoplasmic and nuclear localization of Notch receptors in tumor cells of TNBC patients. It was observed that positive expression of nuclear Notch1 receptor (18%) was found be significantly correlated with high BR score (p=0.009) and necrosis (p=0.003) while cytoplasmic expression of Notch2 receptor (26%) was significantly correlated with local or distant metastasis (p=0.04), worse DFS (p=0.05) and poor OS (p=0.02) in TNBC patients. Further, it was determined that cytoplasmic (65%) and cytonuclear (3%) expression of Notch3 was significantly associated with poorly differentiated tumors (p=0.008) and necrosis (p=0.04) respectively. However, cytoplasmic Notch3 and Notch4 expression were negatively correlated with poor prognostic factors. <jats:bold>Conclusion:</jats:bold> Our data indicated that Notch receptors play a key role in promoting TNBC and mainly, Notch2 receptor may contribute to poor prognosis of the disease. Hence, it is implicated that Notch2 receptor may serve as a potential biomarker and therapeutic target for TNBC.</jats:p>
|
[
{
"section_content": "Breast cancer is the most commonly diagnosed type of cancer, accounting for 24.5% newly registered cases in women and 11.7% newly registered cases in both sexes in a year (https://gco.iarc.fr/today/data/factsheets/populations/900-world-fact-sheets.pdf).Globally, it is second leading cause of death in women after lung cancer (https://gco.iarc.fr/today/data/factsheets/populations/900-world-fact-sheets.pdf).Breast cancer is a phenotypically and biologically heterogeneous disease with diverse response to the treatments offered [1].Immunohistochemical pro ling categorizes breast cancer into four molecular subtypes -luminal A, luminal B, Her2 enriched and Triple negative breast cancer [2].Luminal A, luminal B and Her2 enriched subtypes are characterized by hormone receptor positivity and better prognosis than TNBC.Triple negative breast cancer is de ned by the lack of expression of hormone receptors (ER-, PR-, HER2-), which accounts for 15-20% of breast cancer cases [3,4].TNBC subtype has unfavorable prognosis and patients with TNBC are at greater risk of disease relapse or distant metastasis [5].TNBC is characterized as an aggressive form of cancer which includes features such as higher tumor size, high histological grade, advanced stage and lymph node involvement [6].Currently treatment available for TNBC comprises of surgery, traditional chemotherapy with anthracyclines, taxanes, platinum agents and radiotherapy [7]. Recently, Poly (ADP-Ribose) Polymerase (PARP) inhibitors are used as targeted therapy for TNBC patients with BRCA1/2 gene mutations and programmed death-ligand 1 (PD-L1) positive TNBC patients with locally advanced or metastatic disease are bene ted from PD-L1 inhibitors [8,9].Nonetheless, these therapies are bene cial to a small number of TNBC population. The Notch signaling pathway is a highly conserved pathway which regulates fundamental developmental processes such as cellular differentiation, proliferation, organ formation, morphogenesis and apoptosis [10].The Notch signaling pathway is constitutively activated by the interaction of transmembrane Notch receptors and cell-surface ligands on adjacent cells [11].Upon ligand activation, cascade of events is triggered which includes sequential cleavage by tumor necrosis factor alpha (TNF) converting enzyme (TACE) and gamma-secretase complex, releasing the Notch intracellular domain (NICD).The cleaved NICD translocates to the nucleus and binds with transcriptional regulators such as CBF-1/RBPjk, Suppressor of Hairless, and Lag-1 (CSL), Mastermind-like (MAML), p300 in order to initiate the expression Notch target genes [12,13].Abnormal expression of Notch receptors is known to play a crucial role in malignant transformation in haemopoietic malignancies as well as solid tumors [14].Notch signaling pathway is responsible for normal development of mammary glands and the deregulation of Notch signaling pathway is involved in breast carcinogenesis [15].Studies have frequently shown that Notch1, Notch3 and Notch4 are responsible for aggressiveness of TNBC by contributing to increased cell proliferation, anti-apoptotic activity and stem cell maintenance while few studies have reported tumor suppressive role of Notch2 receptors [16].However, the role of Notch receptors in TNBC is quite debatable.The present study evaluated Notch1, Notch2, Notch3 and Notch4 expression by immunohistochemistry and their clinical signi cance in TNBC. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Materials And Methods",
"section_num": "2."
},
{
"section_content": "One hundred female patients diagnosed with TNBC who had undergone surgery and treatment at the Gujarat Cancer & Research Institute, Ahmedabad during 2014 to 2019 were enrolled in this retrospective study.Detailed clinical history of the patients including age, menopausal status, tumor size, nodal involvement, AJCC TNM stage, histological type, histological grade and treatment offered was noted from the institutional records.Patients who had received chemotherapy or radiotherapy prior to the surgery were excluded from the study.Formalin xed para n embedded (FFPE) blocks were retrieved from archives of Histopathology Department of the institute.This study was approved by Scienti c Review and Ethics committees (EC/BHR/01/2020) of the institute. ",
"section_name": "Patients",
"section_num": "2.1"
},
{
"section_content": "Immunohistochemical analysis was used to detect the presence of Notch1, Notch2, Notch3 and Notch4 receptors in formalin xed para n embedded (FFPE) blocks of TNBC patients.4µm sections from FFPE tumor tissue blocks were taken using a microtome (Leica Biosystems, Germany) and the sections were mounted on 3-Aminopropyl triethoxysilane (APES) coated slides.Immunohistochemistry was performed using autoimmunostainer instrument, Ventana Benchmark XT (Ventana Medical Biosystems, USA) and Ventana reagents according to the manufacturer's protocol.Brie y, the protocol includes depara nization with EZ prep solution, antigen retrieval using cell conditioning solution (CC1) at 95°C, followed by incubation with Ultra view DAB inhibitor for 4 minutes, 100µl of respective primary antibody for Notch1 (Monoclonal, 1:50, Invitrogen) for 32 minutes, Notch2 (Polyclonal, 1:25, Invitrogen) for 60 minutes, Notch3 (Polyclonal, 1:50, Invitrogen) for 60 minutes and Notch4 (Polyclonal, 1:150, Invitrogen) for 32 minutes, incubation with Ultra view Horseradish peroxidase (HRP) multimer for 8 minutes.The detection of antigen-antibody complex was carried out by 3,3'-diaminobenzidine (DAB), counterstaining using haematoxylin for 8 minutes and post counterstaining with bluing reagent for 4 minutes.Ultra view DAB detection kit was used for staining of Notch2, Notch3, Notch4 receptors and Opti view DAB detection kit was used for staining of Notch1.The protocol of Opti view DAB detection kit includes additional incubation with HQ linker for 8 minutes, counterstaining and post counterstaining for 16 minutes and 8 minutes respectively.The slides were later mounted using DPX and xylene. ",
"section_name": "Immunohistochemistry",
"section_num": "2.2"
},
{
"section_content": "Expression of Notch receptors (Notch1, Notch2, Notch3 and Notch4) was assessed by semiquantitative scoring method by employing Immunoreactive scoring (IRS) system.The percentage positive cells were graded as 0(negative staining), 1(<10% positive cells), 2(10% -50% positive cells), 3(51% -80% positive cells) and 4(>80% positive cells).Further, staining intensity of the cells was ranked as 0(negative), 1+ (weak), 2+(moderate) and 3+(strong).The immunoreactive score (IRS) for each case was obtained by multiplying the percentage of positive cells and the staining intensity which gives the range of 0-12.Median score was calculated and cases below the median score were considered as negative or low expression and cases above the median score were considered as over expression. ",
"section_name": "Evaluation of Immunohistochemistry",
"section_num": "2.3"
},
{
"section_content": "Statistical analysis was performed using SPSS 26.0 (SPSS, Inc., Chicago, USA).The Chi-square test was to used determine the association between the expression of Notch receptors and clinicopathological parameters and the association between the Notch receptors.The correlation between the two parameters was determined by Pearson's correlation coe cient (r).Disease-free survival (DFS) time was calculated from date of diagnosis of the patient to date of disease relapse or date of last follow-up. Overall survival time was calculated from date of diagnosis of the patient to last follow-up or date of death due to cancer.Univariate survival analysis was performed by Kaplan-Meier method and signi cance of survival differences was calculated using log-rank test.Multivariate survival analysis was performed by Cox regression model with forward stepwise selection, Wald's statistics and hazard ratio (HR) with 95% con dence interval (CI) were calculated to estimate the prognostic signi cance.P value <0.05 was interpreted as statistically signi cant. ",
"section_name": "Statistical Analysis",
"section_num": "2.4"
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "Notch receptors were found to be localized in membrane, cytoplasm and nucleus of the tumor cells.As shown in gure1, positive expression of Notch1 was observed in 18 patients (18%), Notch2 in 31 patients (31%), Notch3 in 84 patients (84%) and Notch4 in 71 patients of TNBC (71%). ",
"section_name": "Correlation of Notch receptors with clinical and pathological parameters",
"section_num": "3.1"
},
{
"section_content": "Notch1 receptor was found to be predominantly localized only in the nucleus of the tumor cells of 18% patients as represented in Figure2.As shown in table 3, Notch1 nuclear positivity was signi cantly associated with high BR score (27%, 17/62, p=0.009) and necrosis (31%, 13/42, p=0.003) and a trend of association was observed between Notch1 positivity and high histological grade (23%, 15/66, p = 0.08). ",
"section_name": "Notch1 receptor expression",
"section_num": "3.1.1"
},
{
"section_content": "Notch2 was localized in cytoplasm of tumor tissue of 26% patients and in nucleus of 4% patients.In 2% patients, coexistent cytoplasmic and nuclear expression of Notch2 was detected.The incidence of cytoplasmic Notch2 expression was signi cantly higher in patients with local or distant metastasis (47%, 7/15, p = 0.04), while there was no signi cant association observed between nuclear and cytonuclear expression of Notch2 and clinicopathological parameters. ",
"section_name": "Notch2 receptor expression",
"section_num": "3.1.2"
},
{
"section_content": "It was observed that Notch3 receptor was found to be most frequently localized in the cytoplasm (65%), while 4% patients exhibited membrane expression, 13% patients and 3% patients exhibited cytomembrane and cytonuclear expression respectively.Although, membrane Notch3 expression was found in few patients, it was observed that tumors which were poorly differentiated exhibited high membrane Notch3 levels (23%, 15/62, p=0.008).Also, a trend of association was observed between overexpression of membrane Notch3 and metastasis (33%, 05/15, p=0.09).Contrary to this, trend of loss of Notch3 expression was observed in patients with high BR score (76%, 47/62, p=0.06).Moreover, cytoplasmic Notch3 expression was more frequently detected in patients with well differentiated to moderately differentiated tumors (43%, 16/37, p=0.01), low BR score tumors (52%, 14/27, p=0.03), early TNM stage (100%, 02/02, p = 0.05), lymph node negative status (51%, 29/57, p=0.02) and perinodal extension negative status (44%, 35/79, p=0.03).It was observed that cytonuclear Notch3 positivity was strongly correlated with necrosis (07%, 03/43, p=0.04). ",
"section_name": "Notch3 receptor expression",
"section_num": "3.1.3"
},
{
"section_content": "Localization of Notch4 receptor was observed in cytoplasm of 54% cases, cytonuclear expression was observed in 13% cases and cytomembrane expression was observed in 4% cases.Low incidence of cytoplasmic Notch4 expression was detected in younger patients (72%, 36/50, p= 0.02) and necrotic tumors (72%, 31/43, p=0.04).However, cytomembrane and cytonuclear expression of Notch4 did not show any signi cant correlation with clinicopathological parameters.The association between Notch4 and clinicopathological parameters is listed in table8. ",
"section_name": "Notch4 receptor expression",
"section_num": "3.1.4"
},
{
"section_content": "It was observed that expression of Notch2 and Notch3 receptors was signi cantly correlated with expression of Notch4 receptor.Patients with increased levels of Notch2 expression exhibited increased levels of Notch4 receptor (84%, r=0.19, p=0.05) and similarly, higher levels of Notch4 receptor were detected in patients with positive Notch3 expression (76%, r=0.26, p=0.008).In addition, Notch1 expression was strongly correlated with nuclear expression of Notch3 (99%, r = 0.22, p=0.02).Further, it was seen that patients with increased levels of Notch1 receptor had decreased levels of cytoplasmic Notch3 receptor (95%, r=-0.26,p=0.007). ",
"section_name": "Intercorrelation of Notch receptors",
"section_num": "3.2"
},
{
"section_content": "The association between expression of Notch receptors and disease-free survival time and overall survival time of TNBC patients was investigated using Kaplan-Meier survival curves.The mean follow-up time was 37.05 months and maximum follow-up time was 75 months.During this period, 15 (15%) patients had local or distant metastasis and 15 (15%) patients had died due to the disease.With regard to disease-free survival, patients expressing higher cytoplasmic Notch2 (27%, 7/26, 50.71±4.50months) had worse disease-free survival than patients expressing lower cytoplasmic Notch2 (11%, 8/74, 66.83±2.71months, χ 2 = 3.77, df=1, p=0.05).With regard to overall survival, it was observed that patients with higher incidence of cytoplasmic expression of Notch2 (31%, 8/26, 50.60±3.87months) had poor overall survival than patients with low expression of cytoplasmic Notch2 (10%, 7/74, 67.93±2.48months, χ 2 = 5.44, df=1, p=0.02).Expression of other Notch receptors did not show any statistically signi cant relationship with disease-free survival and overall survival. ",
"section_name": "Univariate Survival analysis for expression of Notch receptors in TNBC",
"section_num": "3.3"
},
{
"section_content": "Multivariate survival analysis using Cox regression model with forward selection method (likelihood ratio) revealed that, with regard to disease-free survival advanced TNM stage (Wald = 9.36, HR = 0.19, 95% CI = 0.066-0.551,p = 0.002) entered at step 1 and high BR score (Wald = 7.94, HR = 4.93, 95% CI = 1.626-14.977,p = 0.005) entered at step 2, indicating their role as poor prognostic factors.With respect to overall survival, greater tumor size (Wald = 10.69,HR = 0.57, 95% CI = 0.10-0.321,p = 0.001) entered at step 1, positive lymph node status (Wald = 5.482, HR = 7.209, 95% CI = 1.380-37.676,p = 0.019) entered at step 2, high BR score (Wald = 8.355, HR = 7.787, 95% CI = 1.936-31.317,p = 0.004) entered at step 3 and higher grade (Wald = 8.762, HR = 0.012, 95% CI = 0.001-0.223,p = 0.003) entered at step 4. Multivariate analysis carried out by Cox regression model to evaluate the prognostic role of Notch receptors expression, independent of clinicopathological parameters revealed that with respect to overall survival, cytoplasmic expression of Notch2 (Wald = 4.87, HR = 3.152, 95% CI = 1.138-8.734,p = 0.027) entered at step 1. ",
"section_name": "Multivariate Survival analysis",
"section_num": "3.4"
},
{
"section_content": "Notch signaling pathway is known to play a vital role in normal development and growth.Notch signaling pathway is involved in embryonic development, cellular differentiation, proliferation, homeostasis and apoptosis.Numerous studies have reported that aberrant Notch expression is involved in neoplastic transformation and tumorigenesis.There are four known Notch paralogs which display different patterns in their expression and function across different malignancies.Oncogenic role of Notch signaling was rst reported in T-ALL [17] and later reported in lung cancer, ovarian cancer, breast cancer, colorectal cancer [18,19,20,21,22] while tumor suppressive role of Notch signaling was reported in pancreatic cancer and hepatocellular carcinoma [23,24].However, research about relationship between Notch receptors and TNBC, and the association among the Notch receptors is limited and unclear. In this study, we have examined the role of Notch receptors in TNBC cohort by analyzing the cellular localization of all four known Notch receptors by immunohistochemistry.We observed that Notch1 was expressed in the nucleus of tumor cells suggesting the translocation and accumulation of Notch intracellular domain (NICD1) in the nucleus of tumor cells.The accumulation of NICD1 in the nucleus was strongly correlated with high BR score and necrosis, indicating the tumor aggravating role of Notch1.Our data was in concordance with studies reporting the association of high grade tumors and poor prognosis of TNBC patients with up-regulated Notch1 [25,26,27]. Furthermore, upon examination of Notch2 protein it was revealed that Notch2 was localized in the cytoplasm and nucleus of tumor cells.Despite, various studies showing evidence of tumor suppressive role of Notch2 [28, 29,30], our data suggests that Notch2 receptor plays a tumor promoting role in TNBC.It was observed that high cytoplasmic Notch2 expression was signi cantly correlated with local or distant metastasis, however nuclear and cytonuclear expression of Notch2 did not show any statistical signi cance with clinicopathological parameters.Nonetheless, our study was in line with evidence presented by Lee et al. and Sehrawat et al., stating that inhibition of Notch2 was responsible for antiapoptotic activity and inhibition of migration in TNBC cells [31,32].Also, our study demonstrated that high levels of cytoplasmic Notch2 was positively correlated with worse disease-free survival and poor overall survival proving its tumor promoting role.Earlier studies regarding Notch pathway emphasized that nuclear translocation of NICD was required in order to trigger the downstream target genes.Although, recently it has been demonstrated that in non-canonical Notch pathway, nuclear translocation is not necessary for the pathway to be activated and it may interact with other molecules in the cytoplasm to activate the pathway [33].Therefore, it may be suggested that the association between cytoplasmic Notch2 expression and poor prognosis of the patients might be due to non-canonical Notch pathway.Moreover, our data represents that majority of TNBC samples exhibited high Notch3 protein levels in the cytoplasm.Along with cytoplasmic expression, membrane, cytomembrane and cytonuclear protein expression were also detected.Notably, it was revealed that up-regulated cytoplasmic Notch3 expression in tumors was associated with low BR score tumors, early TNM stage, lymph node negative status and perinodal extension negative status indicating that patients with up-regulated cytoplasmic Notch3 might have favorable prognosis.Our data was in line with studies demonstrating tumor suppressive role of Notch3.Zhang and colleagues observed that high expression of Notch3 inhibited cellular proliferation and tumorigenesis by activating PTEN resulting in better prognosis of breast cancer patients [34].Another study by Chen and colleagues provided evidence that overexpression of Notch3 inhibited cell cycle progression by upregulating levels of Cdh1 in MDA-MB-231 cell lines.However, contrary to above ndings, it was observed that membrane and cytonuclear expression of Notch3 were strongly correlated with poorly differentiated tumors and necrosis respectively.Also, a trend of association was observed between frequent membranous Notch3 expression and metastasis.Although, cytonuclear expression of Notch3 was not detected frequently it was strongly associated with necrotic tumors suggesting the role of nuclear translocation in tumor promoting activity of Notch3.These results were parallel to the ndings of Choy and colleagues that revealed the involvement of nuclear Notch3 expression in tumor promotion [35].The association of increased levels of Notch3 with poorly differentiated tumors might be due to the non-canonical pathway, where membrane-bound Notch3 may have interacted with cell regulators present in the cytoplasm.Contrary to that, membrane Notch3 expression was tended to associate with low BR score tumors.These results of Notch3 suggests that nuclear translocation of Notch3 is potentially involved in promoting tumor aggravating activity, whereas membrane-bound Notch3 may act as an oncogene via activation of non-canonical pathway which does not essentially require nuclear translocation.However, the strong association of high cytoplasmic Notch3 expression with low BR score tumors, early TNM stage, lymph node negative status and perinodal extension negative status might be due the immature state of the receptor or interaction of cytoplasmic Notch3 with tumor suppressing molecules which might be responsible for favorable prognosis of TNBC patients. In addition, in the majority of the patients expressing Notch4 receptor, it was frequently detected in the cytoplasm.This study revealed that high levels of cytoplasmic Notch4 were negatively associated with younger age and necrotic tumors.Contrary, to a study by Wang et al. which stated that Notch4 was associated with poor prognosis for TNBC patients [36], our data represents that Notch4 is inversely correlated to poor prognostic factors of the disease and may function as a tumor suppressor.Concurrently, few studies have presented over expression of Notch4 receptor as tumor suppressive in melanoma and endometrial cancer [37,38]. Further, we evaluated the co-expression patterns of Notch receptors in TNBC patients.It was observed that Notch2 and Notch3 receptors showed positive correlation with Notch4 receptor.This may be interpreted as Notch receptors are signi cantly associated with each other and even though Notch4 might function as a good prognostic factor, together with Notch2 and Notch3 receptors they contribute to tumor promotion and poor survival of TNBC patients.Moreover, it was observed that nuclear expression of Notch3 was positively associated with nuclear expression of Notch1 suggesting the oncogenic role of nuclear localization of both Notch1 and Notch3 receptors.Additionally, it was noted that nuclear Notch1 expression was negatively correlated with the cytoplasmic Notch3 expression indicating opposing role of both the receptors based on their localization, as cytoplasmic Notch3 is associated with better prognosis and nuclear Notch1 is linked to poor outcome of the disease.Therefore, our results imply that interplay of Notch receptors is rather complex mechanism. In conclusion, our study demonstrated that Notch receptors exhibit a pleiotropic role in TNBC.Notch1 and Notch2 receptors play a tumor promoting role and contribute to poor outcome in TNBC patients.It was also revealed that although membrane and cytonuclear Notch3 had tumor aggravating effects, cytoplasmic Notch3 and Notch4 receptor had tumor suppressive effect.It is implicated that the tumor promoting activity of Notch receptors might be due to the canonical Notch signaling pathway which essentially requires the nuclear translocation of NICD to trigger the transcriptional regulation of target genes or it might be due to the non-canonical pathway which is activated through the interaction of Notch receptors with transcriptional regulators present in the cytoplasm to exert its effects.The tumor suppressive effect of cytoplasmic Notch3 and Notch4 expression may be due to the inactive state of the receptor or its interaction with tumor suppressive molecules in the cytoplasm.Therefore, it is important to assess the subcellular localization of the Notch receptors as they are ubiquitously distributed in the cells in order to evaluate their individual effects in TNBC.Hence, our results strongly indicate that Notch receptors are involved in promoting TNBC and chie y, Notch2 receptor may serve as a potential prognostic marker and therapeutic target for patients with TNBC. ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "",
"section_name": "Statements And Declarations",
"section_num": null
}
] |
[
{
"section_content": "Acknowledgments: This research was supported by The Gujarat Cancer Society and The Gujarat Cancer & Research Institute. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "This work was supported by The Gujarat Cancer Society (GSC), Ahmedabad, Gujarat, India under grant number EC/BHR/01/2020. The authors have no con ict of interest. ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "This work was supported by The Gujarat Cancer Society (GSC), Ahmedabad, Gujarat, India under grant number EC/BHR/01/2020. ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "The authors have no con ict of interest. ",
"section_name": "Con ict of Interest",
"section_num": null
},
{
"section_content": "Data and material of this study may not be available for another purposes. ",
"section_name": "Availability of Data and Material",
"section_num": null
},
{
"section_content": "All authors contributed to the study conception and design.Material preparation, data collection and analysis were performed by Ms. Heer Shah with the help of Ms. Mittal Mistry.The rst draft of the manuscript was written by Ms. Heer Shah under observation of Dr. Hemangini Vora and all authors commented on previous versions of the manuscript.All authors read and approved the nal manuscript. Institute and The Gujarat Cancer Society (EC/BHR/01/2020), Ahmedabad, Gujarat, India on 23/03/2020. Informed consent was obtained from all individual participants included in the study. The authors a rm that all the participants in the study provided informed consent for publication of their individual data including medical history and treatment. Relationship between disease-free survival and Notch2 Relationship between overall survival and Notch2 ",
"section_name": "Author Contributions",
"section_num": null
},
{
"section_content": "All authors contributed to the study conception and design.Material preparation, data collection and analysis were performed by Ms. Heer Shah with the help of Ms. Mittal Mistry.The rst draft of the manuscript was written by Ms. Heer Shah under observation of Dr. Hemangini Vora and all authors commented on previous versions of the manuscript.All authors read and approved the nal manuscript. ",
"section_name": "Author Contributions",
"section_num": null
},
{
"section_content": "",
"section_name": "Ethics approval",
"section_num": null
},
{
"section_content": "Institute and The Gujarat Cancer Society (EC/BHR/01/2020), Ahmedabad, Gujarat, India on 23/03/2020. ",
"section_name": "This study was approved by Scienti c Review and Ethics committees of The Gujarat Cancer & Research",
"section_num": null
},
{
"section_content": "Informed consent was obtained from all individual participants included in the study. ",
"section_name": "Consent to participate",
"section_num": null
},
{
"section_content": "The authors a rm that all the participants in the study provided informed consent for publication of their individual data including medical history and treatment. Relationship between disease-free survival and Notch2 Relationship between overall survival and Notch2 ",
"section_name": "Consent to publish",
"section_num": null
}
] |
10.21203/rs.3.rs-2244003/v1
|
Cycloserine-induced psychosis in patients with drug-resistant tuberculosis: a systematic review of case reports
|
<jats:title>Abstract</jats:title> <jats:p>Objectives To describe the clinical characteristics and outcomes of cycloserine (CS) - induced psychosis in adults diagnosed with drug-resistant tuberculosis (DR-TB). Materials and Methods A systematic review of case reports was carried out according to PRISMA guidelines. Subsequently, information was extracted concerning sociodemographic variables, clinical characteristics of psychosis, treatment, and clinical outcomes, as well as the quality of the articles using a standardized tool (Joanna Briggs Institute -JBI- Case Reports Tool). Results Of 3416 articles, 20 reports from seven countries were included, encompassing 22 patients (68.18% male participants, mean age: 31.45 ± 10.88 years). Delusions (68.20%, primarily paranoid) were the most frequent psychotic symptom. The median duration of the psychotic episode was 13 days (interquartile range: 35). Other frequently appearing symptoms in CS-induced psychosis were aggressiveness (68.20%), insomnia (59.10%), hallucinations (54.50%), incoherent/disorganized speech (45.45%) and irritability (45.45%). After antipsychotic treatment (81.81% of the reported cases were treated with at least one antipsychotic), 95.50% presented improvement, while 4.54% died by suicide. Finally, after the quality assessment of studies using the JBI tool, 85.00% of the articles showed a low risk of bias. Conclusions CS-induced psychosis is a rare presentation, generally of short duration, that includes delusions (mostly paranoid) as its main psychotic symptom and shows mostly a symptom improvement after medical treatment. PROSPERO registration number: CRD42022359551 (Date of registration: 22/09/2022)</jats:p>
|
[
{
"section_content": "Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis, currently having one of the highest mortality rates (1)(2)(3) and causing the death of approximately 1.3 million people worldwide (4-6).In the last decades, a growing issue concerning this infectious disease is the increasing number of cases resistant to rst-line antibiotics (i.e., rifampicin and isoniazid), which also represents higher costs for health services and a higher burden of disease (7,8).As a solution to the treatment of TB-resistant patients, evidence-based guidelines and world medical societies contemplated different treatment options, including extended-spectrum antibiotics with robust effectiveness, such as cycloserine (D-4amino-3-isoxazolidine) (9)(10)(11).The latter competitively inhibits two essential enzymes for the synthesis of the cell wall of Mycobacterium tuberculosis, exerting its antibiotic effect (12,13).However, the therapy with cycloserine (CS) also correlates with the appearance of psychotic symptoms since CS can penetrate the brain-blood barrier, inhibits GABA-transferase, and interacts with N-methyl-D-aspartate (NMDA) receptors in the central nervous system (14)(15)(16).Although CS may induce psychosis during treatment, its appearance is considered a non-frequent condition among drug-resistant (DR) TB patients (17)(18)(19).For instance, a longitudinal study reported that in a group of 144 patients with multidrug-resistant (MDR-TB) treated with CS, four patients (8%) developed psychosis (12). Moreover, a pharmacological safety and surveillance study estimated that the global combined prevalence of adverse drug reactions due to CS was 9.0%, and 5.7% for psychiatric disorders (17). Additionally, of the 445 safety reports of cases reported since 1970 attributed to CS, 75% were attributed to neuropsychiatric conditions, and of these, 14% related to psychosis (17), considered in this study also a not very frequent condition.Since the appearance of induced psychosis during a CS treatment is very low, most published studies concerning cycloserine-induced psychosis are restricted to case reports. Although case reports are unsuitable for inferring causality and cannot be extrapolated, they often provide essential aspects missing in population studies, especially in rare conditions (20), such as cycloserine-induced psychosis.In this sense, conducting a systematic review of case reports would help summarize clinical and therapeutical aspects of CS-induced psychosis in MDR-TB over time (20)(21)(22)(23). Therefore, the main objective of this study is to perform a systematic review to describe the main clinical characteristics, comorbidities, therapy, and clinical outcomes of the cases reported in the literature on cycloserine-induced psychosis in DR-TB adult patients.In addition, we excluded studies if the reported participants were under 18 years, did not have TB, or were not in treatment with CS.Furthermore, we excluded studies with patients with a previous history of schizophrenia spectrum disorders or bipolar disorder, with illegal substance misuse during CS treatment, and with a current disease that could explain the induced psychosis (e.g., cerebral tumor or stroke) during the CS treatment.Studies that were not case report articles, case series articles, or manuscripts describing TB patients with CS-induced psychosis were excluded.Finally, articles that were not available as full-text were also excluded. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials And Methods",
"section_num": null
},
{
"section_content": "Firstly, a systematic literature search was conducted between 07/23/2022 and 08/24/2022 in different scienti c literature databases, including MEDLINE (PubMed, National Center for Biotechnology Information, National Library of Medicine, United States of America), EBSCO (EBSCO Industries Incorporate, United States of America), Web of Science (Clarivate Analytics, United States of America), CENTRAL (Cochrane Central Register of Controlled Trials, Cochrane Library, United States of America), SciELO (Scienti c Electronic Library Online, Brazil), and Google Scholar (Google Incorporate, United States of America).In each case, the literature search was carried out by combining different Boolean operators (e.g., \"psychosis\", \"tuberculosis\"), having the word \"cycloserine\" in the operator.More details concerning the systematic literature search, combination of Booleans, and the Boolean formulae are described in supplement material 1. Finally, between 10/03/2022 and 10/05/2022, the search for articles was conducted again in the databases mentioned above using the same search strategy.We found 21 additional articles (3437 articles, supplemental material 1); however, the additional articles were not relevant publications to this systematic review or they not ful ll the inclusion criteria, not affecting the results that we found, concluding the search on 10/05/2022. ",
"section_name": "b. Search strategies",
"section_num": null
},
{
"section_content": "Posteriorly, the literature search results were extracted as .csv,.ris,or .txtles, depending mainly on the search platform and database used.In the case of the search platform Google Scholar, we used the free access software Publish or Perish (24,25) to extract the results of the systematic literature search in the formats mentioned above.After extraction, all obtained data les were imported into the free access software Zotero v.6.09(Corporation for Digital Scholarship, United States of America) to remove duplicated articles or records. Afterward, we exported the remaining data without duplicates in the web application Rayyan.ai(26), where the authors ACS, SVQ, and LUQ independently selected all case reports, case series, and correspondence using the title or abstract related to cycloserine-induced psychosis.Disagreements in the articles' selection were resolved by consensus among all the authors.Later, each author performed a screening of the articles to exclude all those articles that did not meet the inclusion criteria.Likewise, the disagreements concerning inclusion and exclusion criteria were resolved under the consensus modality.Neither the year of publication of the articles nor their language was an exclusion criterion.In case of possible language issues, we used a free access translator (Google Translate, Google Inc., United States of America) to obtain the main information of the manuscript. ",
"section_name": "c. Selection process",
"section_num": null
},
{
"section_content": "The evaluation of the risk of bias and the quality assessment was carried out using The Joanna Briggs Institute (JBI) Critical Appraisal tool for use in Systematic Reviews (27), which was designed to evaluate the methodological quality and the risk of bias in case report studies (28-31). The remaining articles were randomly assigned to three authors (ACS, SVQ, and LUQ).During the quality assessment, each author could not reveal to the other colleagues the title or content of randomly assigned articles to make the quality assessment procedure as blind as possible.Authors were also asked not to ask for help or support from other authors while reading and assessing the quality of the assigned case reports. If the articles did not meet the criteria for a low risk of bias described in the JBI tool, the article was sent to a second reviewer for evaluation before exclusion.In this case, the second reviewer received the article without any previous assessment or judgment of the rst one to avoid bias at the moment of the second quality assessment.Finally, a third reviewer determined the article's inclusion or exclusion in case of discrepancies between the rst and the second evaluators.For this purpose, we established a structure for the quality assessment in the following order ( rst-second-third reviewer): ACS-SVQ-LUQ, LUQ-ACS-SVQ, SVQ-LUQ-ACS. ",
"section_name": "d. Quality of studies",
"section_num": null
},
{
"section_content": "A frequent limitation of case reports is to establish causality in adverse drug reactions, and this is due to different factors, such as the subjectivity of the case report and speci c characteristics of the patient.One way to deal with these limitations and standardize the information presented is through the Naranjo algorithm (Naranjo Adverse Reaction Probability Scale) (32).This tool is widely used in case reports and consists of 10 criteria with which a score is assigned that determines one of the four categories: doubtful (0 points), possible (1 to 4 points), probable (5 to 8 points), and de nite (≥ 9 points) (33). If the included case report article did not report the scores following the Naranjo algorithm, we used the information described in the case reports to estimate the category corresponding to the Naranjo algorithm, as also recommended in other studies (34). ",
"section_name": "e. Naranjo Adverse Reaction Probability Scale",
"section_num": null
},
{
"section_content": "Concerning the data extraction, three authors (ACS, SVQ, LUQ) independently carried out this process using a template programmed for the study in Microsoft Excel.Again, all included articles were randomly assigned among these three authors, collecting and registering information concerning (1) the year of publication, (2) patients' sociodemographic characteristics (e.g., sex, age, and country), (3) clinical characteristics of the CS-induced psychosis, (4) treatment used against the psychotic episode (e.g., antipsychotic treatment, CS treatment discontinuation, CS dose reduction or maintenance), and (4) clinical outcomes of the CS-induced psychosis. ",
"section_name": "f. Data extraction",
"section_num": null
},
{
"section_content": "Since this systematic review sought mainly to describe the characteristics of CS-induced psychosis in the literature, we applied descriptive statistics to present the most frequent symptoms of CS-induced psychosis and the sociodemographic characteristics and treatment used.For this purpose, we presented the quantitative data using the appropriate measures of central tendency (mean with standard deviation and median with interquartile range).Qualitative data, mostly dichotomous, were expressed using percentages and frequencies.If needed, data were presented in tables and graphs for better readability.Concerning data analysis, SPSS software (International Business Machines Corporation, New York, United States of America), version 26.0, was used for the descriptive data. ",
"section_name": "g. Statistical analysis",
"section_num": null
},
{
"section_content": "Figure 1 shows the ow diagram according to the PRISMA protocol for the studies included in this systematic review of case reports.Initially, 3416 articles were identi ed; 1837 duplicates were eliminated, and 1579 publications remained.Subsequently, 1531 articles were removed after reviewing the titles and abstracts because of irrelevance to the study objective.Of the remaining 48 articles, 24 could not be retrieved and were excluded, and 3 articles were excluded since the reported patients were underage. Finally, 21 articles were assessed with the JBI tool after the screening.One article (35) was excluded from the quality assessment process due to the low quality of information presented and the high risk of bias. A total of 20 articles (36-55) met the inclusion criteria and survived the quality process, encompassing 22 patients from 8 different countries (Table 1) and most patients from India (10 patients, 45.45% of the cases included).One of the articles was published in Spanish (53) and another in Japanese (37), while the rest were published in English. Of the 22 patients included in the case reports, 15 (68.18%) were male, and 7 (31.82%)were female patients.Additionally, the mean age (standard deviation) among the participants was 31.45 (10.88) years. Regarding past medical history, 15 (68.18%) had no medical conditions.However, 2 (9.09%) patients had diabetes mellitus as comorbidity, 1 (4.54%) had hypothyroidism, 1 (4.54%) had an episode of meningeal tuberculosis in the past, and 1 (4.54%) had suicide attempts during TB therapy.In this patient's case, the study authors reported no relevant psychiatric history.Of the included studies, one patient had a family history of psychiatric disorders (major depression and bipolar disorder), and one patient had a family history of alcoholism; however, there were no reports of medical comorbidities for both patients.Finally, 19 (86.36%) reported having no illegal drug, alcohol, or nicotine dependency.However, 3 (13.64%)participants reported having used alcohol regularly in the past, and one (4.54%)patient reported the last use of cannabis and cocaine 9 weeks before admission.No reported patient consumed concomitantly illegal drugs or alcohol during the treatment with CS. b. Clinical characteristics of DR-TB patients and CS-induced psychosis Concerning the type of DR-TB patients, 21 of 22 patients (95.45%) had multidrug-resistant TB (MDR-TB), and one patient (4.54%) had renal tuberculosis (Table 1).Moreover, there were no patients with extreme drug-resistant TB (XDR-TB).Concerning the CS therapy, the mean dose (standard deviation) of CS given as treatment was 631.58 (174.17)mg/die, and the mean duration of CS treatment (standard deviation) until the onset of psychotic symptoms was 169.09 (239.52)days.Within the antitubercular treatment, the reported patients included in this systematic review received mostly drugs, such as pyrazinamide (14 patients, 63.60%), levo oxacin (12 patients, 54.50%), ethionamide (12 patients, 54.50%), kanamycin (11 patients, 50.00%) and pyridoxine (10 patients, 45.50%) (Table 1). Table 1 also mentions the details of the clinical presentations of the patients in the included case reports with MDR-TB and cycloserine-induced psychosis.Regarding the duration of psychosis, the median duration of psychosis during treatment with cycloserine was 13 days (39 - 4 days; interquartile range: 35).The most common symptoms reported in patients with MDR-TB and cycloserine-induced psychosis were delusions (15 patients; 68.20%), followed by aggressiveness (15 patients; 68.20%), insomnia/decreased sleep (13 patients; 59.10%), hallucinations (12 patients; 54.50%), irritability (10 patients; 45.50%) and incoherent or disorganized speech (10 patients; 45.50%).Concerning the aggression observed in the case reports, 14 of 22 reported patients presented aggressive behavior, mostly against other persons, while one of the 22 included patients presented self-aggression (Table 2).In this last case, the patient died as a result of suicide due to exsanguination as a cause of self-injury cuts in the peripheral vessels.In the case of the registry of the type of hallucinations, there are records of 12 (54.50%)patients reported in the included clinical cases.Of these, two (9.10%) of the patients presented visual hallucinations, one (4.54%)presented auditory hallucinations, ve (22.70%) a combination of both, and in four cases (18.20%), the hallucination type was not speci ed (Table 2). Concerning the type of delusions, 7 of 9 patients (77.80%) reported persecutory/paranoid delusions, one of 9 patients (11.11%) reported delusions of grandeur and 1 of 9 patients (11.11%) reported jealous delusions.Finally, 6 patients reported two delusional symptoms (Table 2).Of them, four of 6 patients (66.67%) reference and persecutory/paranoid delusions, and two of 6 patients (33.33%) had persecutory/paranoid and delusions of grandeur (Table 2). ",
"section_name": "Results a. General characteristics of the included reports and patients",
"section_num": null
},
{
"section_content": "Treatment of cycloserine-induced psychosis varied between reported patients.Of the 22 patients reported, only 4 (18.20%) did not receive antipsychotic treatment.Of the remaining 18 (81.80%)patients reported, the majority (10 patients; 45.50%) received monotherapy with one antipsychotic (2 participants received risperidone, four olanzapine, two haloperidol, 1 received quetiapine, and 1 received chlorpromazine).Of the patients who received a combination of 2 antipsychotics (4 patients, 18.20%), two received a combination with olanzapine and haloperidol, 1 received chlorpromazine and haloperidol, and one patient received olanzapine and quetiapine.Finally, four patients (18.20%) received a combination of three antipsychotics: 2 of them received chlorpromazine + risperidone + haloperidol, 1 of them received haloperidol + promethazine + olanzapine, and 1 of them received haloperidol + chlorpromazine + olanzapine. Of the adjuvant treatments to antipsychotic therapy, six (27.20%) patients received benzodiazepines (lorazepam, nitrazepam, and clonazepam), 3 (13.60%)patients received anticholinergic agents (benzhexol), and 1 (4.54%) received valproic acid. Parallel to psychopharmacological therapy, 11 (50.00%) of the reported patients stopped receiving CS, while 2 (9.19%) continued CS treatment, and 2 (9.19%) reported patients the dose of CS was reduced (Table 1).Additionally, two reported patients (9.19%) had their cycloserine dose wholly suspended for a de ned period (Table 1).Finally, in 3 (13.60%)reported cases, the antitubercular therapy (including CS) was withheld (Table 1). Finally, 20 of the 22 reported patients (90.90%) presented a clinical improvement of psychotic symptoms with the reported therapeutic strategies.On the other hand, only one patient reported partial symptom improvement with low-grade labile mood and psychosis (Table 1).Finally, in the case of one patient, suicide was committed by exsanguination by cutting himself in different regions of the body and did not present an improvement in psychotic symptoms despite treatment (Table 1). ",
"section_name": "c. Clinical treatment of MDR-TB patients with CS-induced psychosis",
"section_num": null
},
{
"section_content": "In general, the quality of the case reports was good, showing that the vast majority of the articles had a low risk of bias (supplement material 2).In this sense, it was determined that 17 (85.00%) of the reports presented a very low risk, while 3 (15.00%)presented a low risk.A total of 3 (15.00%)studies did not report the number of doses of cycloserine used in patients with MDR-TB.On the other hand, in the case of the record of symptoms, of the reported cases that described the presence of hallucinations, 4 (20.00%)studies did not specify what type of hallucinations they were (e.g., visual or auditory).Finally, only one study described the use of cycloserine for treating MDR-TB after using other antibiotics.At the same time, the rest of the reported cases indicate a concomitant use of cycloserine with other antibiotics.The quality assessment of the individual studies is in detail in supplement material 2. In the case of the probability of the reaction to an adverse event, in this case, psychosis induced by cycloserine, a total of 6 articles used the Naranjo algorithm in patients who use cycloserine.In case this algorithm had not been described in the case reports, the score was established according to the information presented in the article.Of the reported articles, 18 showed a \"probable\" Naranjo index for cycloserine-induced psychosis (5 to 8 points), while the rest scored as \"possible\" (1 to 4 points). ",
"section_name": "d. Quality assessment of case reports: CS-induced psychosis in DR-TB patients",
"section_num": null
},
{
"section_content": "The following systematic review of case reports identi ed 20 articles (years of publication between 1965 and 2022) concerning CS-induced psychosis in DR-TB patients (mean age: 31.45 years), whose majority did not present comorbidities.Furthermore, the median duration of the CS-induced psychosis was 13 days, and the most prevalent psychotic symptom among the patients was delusions (mostly paranoid or persecutory).Concerning the antipsychotic treatment against CS-induced psychosis, it was found that most received at least one antipsychotic, and parallelly CS treatment was stopped.Of the reports included in this review, 20 of 22 patients showed a clinical improvement.However, only one reported patient (treatment: risperidone and clonazepam) committed suicide by in icting himself on multiple cuts and therefore dying of exsanguination.Finally, most of the included case reports showed a very low risk of bias at the time of evaluation; also, the Naranjo index showed psychosis as a \"probable\" adverse reaction of CS in most of the reports. Regarding the sociodemographic data, there are similarities between our ndings and previous studies with CS-induced psychosis.For example, a retrospective study reported that younger ages represented a risk factor for CS-induced psychosis (56).In another observational study, a mean age of 35.7 years was reported in a sample of 144 patients, as well as a predominance of males in the sample, similar to the sample characteristics of our study (12). To the best of our knowledge, this is the rst systematic review of case reports which describes the psychopathology of CS-induced psychosis in reported DR-TB patients.Our results showed that paranoid delusions and delusions of persecution are the most frequent psychotic symptoms among the reported cases.Other studies with experimental designs, for instance that used CS as adjuvant treatment for eight weeks in schizophrenic patients, also demonstrated a frequency increase in delusions, grandiosity, and hostility (57).Finally, similar results are reported in an experimental study that found an exacerbation of psychotic symptoms through CS in a small group of patients with schizophrenia (hallucinations, an acute paranoid delusion, psychomotor agitation, thought disorganization, and catatonic symptoms) or caused a confusional psychosis (mainly including an obnubilation of consciousness and formal thought disorders with speech acceleration, that occur especially in intoxications) with circadian disorders (58). Currently, some studies support the role of CS as a partial agonist of NMDA receptors binding to glycine sites (57,59).However, at high doses, CS can act as NMDA receptor (NMDA R ) antagonists (60, 61), generating or worsening psychotic symptoms, as reported, for instance, in patients with schizophrenia (57,62).In addition, the dose-dependent NMDA antagonism could also explain the induced psychosis of the reported patients with DR-TB, receiving CS doses between 250 and 1000 mg.However, an observational study found that CS concentrations and the area under the curve were not associated with the appearance of psychotic symptoms in patients with MDR-TB (12), remaining unclear the pharmacological mechanisms of CS psychosis induction in patients with DR-TB. Regarding the treatment of CS-induced psychosis, most reported patients presented an improvement with the administration of antipsychotic agents and the discontinuation of CS.This characteristic varies according to the literature reports since a retrospective study shows the improvement of psychosis in most patients by reducing or temporarily suspending the CS dose (56).However, the results of both studies agree that a reduction in CS exposure and the administration of antipsychotics reduces the frequency of the appearance of psychotic symptoms produced by CS. Finally, the Naranjo adverse reaction scale showed that the induction of psychosis due to CS is probable (5 to 8 points).Some case reports presented values on the Naranjo scale (6 articles); however, the remaining reports did not mention a score in the Naranjo algorithm, which the authors of this study nally calculated.Naranjo's tool may have some disadvantages, among which is the variability of the numerical score due to the evaluator's opinion.In addition, the lack of information described in the article to qualify certain criteria could make it di cult to assess the probability of an adverse reaction in the case, for example, of external evaluators who were not involved in the treatment of the patient.This di culty in reproducibility has been analyzed in other studies by comparing the score assigned by the same authors of the case report and that obtained by evaluators only with the information provided by the article (34). In this case, it is observed that despite variation in the numerical score, this did not in uence a signi cant change in the assigned category (34). The main strength of the present study includes a description, through a systematic search, of psychotic symptoms in patients who use CS due to DR-TB since psychosis due to CS is less frequent (between 8% and 14%) (12,17). However, it is important to mention that this study has limitations that must be considered.First, the small sample size of this systematic review.Secondly, some reports presented incomplete data (e.g., the characterization of hallucinations or the dose of CS).In addition, ve articles did not present auxiliary tests (routine laboratories, diagnostic imaging, electrophysiology, etc.).Regarding the study's methodology, this systematic review of case reports is based on non-systematized clinical information, which in uenced the presentation of the details and the quality of the articles included, being a limitation of the study.Likewise, this study is not appropriate to determine the causality of the psychotic event due to the non-randomized and anecdotal methodology of the clinical cases.Finally, the accessibility of some articles was very restricted, despite contacting the corresponding authors on multiple occasions, in such a way that in these cases, only the abstract of the case report or the title of the report was available. In light of our results, we conclude that the most frequent psychotic symptom of CS-induced psychosis was paranoid delusions or delusions of persecution.In addition, CS-induced psychosis is of short duration that shows mostly a symptom improvement after medical treatment, involving the CS withhold/suspension and antipsychotic treatment.Future studies should clarify possible associations between psychotic symptoms and CS serum concentrations and observe the risk factors associated with the development of psychosis due to CS in patients with DR-TB. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "Con ict of interests: The authors declare no con ict of interests. This is a list of supplementary les associated with this preprint.Click to download. ",
"section_name": "Declaration",
"section_num": null
},
{
"section_content": "Con ict of interests: The authors declare no con ict of interests. ",
"section_name": "Declaration",
"section_num": null
},
{
"section_content": "This is a list of supplementary les associated with this preprint.Click to download. ",
"section_name": "Supplementary Files",
"section_num": null
}
] |
10.7759/cureus.50801
|
Corneal Copper Deposition Secondary to Monoclonal Gammopathy in a Patient With Chronic Lymphocytic Leukemia: A Case Report
|
Hypercupremia-induced corneal copper deposition secondary to monoclonal gammopathy is rare and shows a characteristic corneal opacity quite different from other causes of hypercupremia, such as Wilson's disease. This report describes a case of corneal copper deposition in a patient with monoclonal gammopathy associated with chronic lymphocytic leukemia. An 84-year-old man with slowly progressive corneal opacity was referred to our hospital. The corneal opacity was present at least five years ago. The patient's best-corrected visual acuity was 20/25 OU (in both eyes) at the initial visit to our hospital. Slit-lamp examination and anterior segment optical coherence tomography revealed bilateral brown-colored opacity localized to deep layers of the central cornea. In vivo confocal microscopy (IVCM) showed indistinct corneal stromal cells in the deep layer and endothelial cells. The possible differential diagnoses were corneal dystrophy and Wilson's disease, but the color, shape, or site of corneal opacity was inconsistent with the disease. As the patient had a history of chronic lymphocytic leukemia, which is often associated with monoclonal gammopathy, we suspected that the corneal opacity was copper deposition in association with the hematologic diseases. Laboratory examinations showed elevated serum copper and normal ceruloplasmin. Serum protein electrophoresis revealed significantly high IgG levels with depression of IgA, IgE, and IgM. These results supported our diagnosis. Followingly, we consulted the patient's attending hematologist, and the doctor initiated treatment for hypercupremia. In conclusion, hypercupremia secondary to monoclonal gammopathy should be considered a possible cause of central brown-colored corneal opacity.
|
[
{
"section_content": "Hypercupremia can lead to corneal copper deposition, typically associated with the peripheral Kaiser-Fleischer ring in Wilson's disease [1].This report focuses on an unusual presentation of corneal copper deposition: bilateral brown-colored corneal opacity in the central cornea.The case involves a patient with chronic lymphocytic leukemia (CLL) and associated monoclonal gammopathy.Several reports have shown that monoclonal gammopathy can cause central copper deposits, but such cases are quite rare [2][3][4][5][6][7]. CLL, often linked to monoclonal gammopathy [8], is central to our exploration of this unique corneal manifestation.The rarity of this presentation prompts deeper investigation into the interplay between hypercupremia, monoclonal gammopathy, and ocular manifestations.By highlighting this atypical case, we aim to contribute to understanding ocular complications in hematologic disorders and help ophthalmologists diagnose the rare pathology. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "An 84-year-old Japanese man with slowly progressive corneal opacity was referred to Hiroshima University Hospital.The patient's corneal opacity was present at the initial visit to the former ophthalmologist five years ago.The patient's medical history included type two diabetes mellitus, hyperuricemia, cholangiocarcinoma after pancreatoduodenectomy, and chronic lymphocytic leukemia (CLL).The patient used to be treated with rituximab for CLL and has been in remission without any treatment for over nine years.The patient's family history was negative for any ocular disease.The patient's best-corrected visual acuity was 20/25 OU at the initial visit to our hospital.The intraocular pressure was 13 mmHg in the right eye (OD) and 14 mmHg in the left eye (OS).Slit-lamp examination showed round and brown-colored opacity at the central and deep cornea in both eyes (Figure 1).Pigmentation also appeared on the central iris.The dense corneal opacity made it difficult to visualize the details of the lens.Anterior segment optical coherence tomography detected hyperreflective materials at the deep layer of the cornea (Figure 2).In vivo confocal microscopy (IVCM) showed indistinct corneal stromal cells in the deep layer and endothelial cells.In contrast, epithelial and stromal cells in the shallow layer were normal and clearly captured.IVCM did not find any deposits (Figure 3).Posterior segment ocular examination was unremarkable.Laboratory examinations showed elevated serum copper (630 μg/dL) and normal ceruloplasmin (20 μg/dL), alanine aminotransferase, and aspartate aminotransferase.Serum protein electrophoresis revealed a high immunoglobulin G (IgG) level of 3,172 mg/dL with depression of IgA, IgE, and IgM.The characteristic ocular findings and the results of systematic examinations suggested that the corneal opacity was copper deposition secondary to hypercupremia associated with IgG monoclonal gammopathy.Followingly, we consulted the patient with the attending hematologist and initiated the treatment of hypercupremia with zinc acetate dihydrate. ",
"section_name": "Case Presentation",
"section_num": null
},
{
"section_content": "Bilateral brown-colored opacity in the central cornea is significantly characteristic of copper deposition secondary to hypercupremia associated with monoclonal gammopathy.However, this condition may not be well-recognized due to its low frequency.We could not make the correct diagnosis immediately in this case.We first considered the possibility of Schnyder corneal dystrophy (SCD).The shape and site of corneal opacity evoked SCD, but the corneal opacity of SCD appears in shallower layers than in our case and is not pigmented.Besides, phospholipids and cholesterol should be detected as spindle-shaped deposits by IVCM in SCD [8].Another differential diagnosis was Wilson's disease.The brown-colored opacity at Descemet's membrane in our case was similar to Wilson's disease, but its site was the opposite.Besides, laboratory data of our patient, normal ceruloplasmin and normal hepatic function, were not consistent with Wilson's disease [1].We also noted the patient's medical history of pancreatoduodenectomy as a possible cause of hypercupremia or corneal copper deposition, but there was no apparent relation between them in literature to the best of our knowledge.Finally, as monoclonal gammopathy is known to be associated with CLL [9], the patient's medical history allowed us to make the proper diagnosis in this case. Shah et al. reviewed eight cases of corneal copper deposition secondary to hypercupremia associated with monoclonal gammopathy, including three multiple myeloma cases and five monoclonal gammopathies of unknown significance [4].Whether the hematologic tumor is malignant or not, monoclonal gammopathy can cause hypercupremia.Bilateral brown-colored corneal opacity at the central Descemet's membrane was observed in all reviewed cases.Corneal transplant and cataract extraction were performed in some cases, revealing the presence of a pigmented band at the Descemet membrane.This was confirmed as copper deposition through Rhodanine staining and X-ray microanalysis [3,4].Besides, although the detail of the lens surface was not visualized as satisfactory in our case due to its dense corneal opacity, some case reports showed that pigment also appeared on the lens capsule and was confirmed as a copper deposit pathologically [3,4]. Elevated IgG, which has a high affinity to plasma copper, is suggested to play an important role in the mechanism for why copper deposits accumulate at the central cornea in patients with monoclonal gammopathy [3].While copper cannot penetrate the anterior chamber unless the blood-aqueous barrier breaks down, IgG can move into the anterior chamber [10] and thus may transfer plasma copper there.However, since excess copper from the bloodstream is believed to penetrate the anterior chamber and accumulate at the Descemet's membrane in Wilson's disease, the hypothesis may not safely explain why copper accumulates in different sites between monoclonal gammopathy and Wilson's disease [11].Additionally, while acknowledging that excess IgG is a crucial factor in the characteristic corneal opacity observed in patients with monoclonal gammopathy, it should be noted that the immunoglobulin is present throughout all areas of the cornea, both centrally and peripherally, at similar levels [12].Therefore, further investigation still seems necessary to comprehend the mechanism of the characteristic copper-deposition pattern in monoclonal gammopathy. One obvious limitation of this case report is that we did not conduct a histopathological examination because the patient did not hope to receive surgical treatments.Nonetheless, given that the patient's corneal opacity was quite distinct and laboratory examinations were consistent with hypercupremia secondary to monoclonal gammopathy, we believe the lack of histopathological examination was not critical to diagnose. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Hypercupremia secondary to monoclonal gammopathy should be considered a possible cause of bilateral brown-colored opacity in the central cornea.In this case, monoclonal gammopathy and hypercupremia had not been mentioned prior.Therefore, it is clinically meaningful that the ocular findings led to treating systematic diseases that would cause various systematic disorders. ",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "All authors have reviewed the final version to be published and agreed to be accountable for all aspects of the work. Acquisition, analysis, or interpretation of data: Koichiro Shinji, Tai-ichiro Chikama, Atsuhiko Fukuto, Yoshiaki Kiuchi, Taka-aki Moriguchi Critical review of the manuscript for important intellectual content: Tai-ichiro Chikama, Atsuhiko Fukuto, Yoshiaki Kiuchi, Taka-aki Moriguchi Supervision: Tai-ichiro Chikama, Yoshiaki Kiuchi Human subjects: Consent was obtained or waived by all participants in this study.the Institutional Review Board of Hiroshima University issued approval E-709.Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work.Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work.Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. ",
"section_name": "Additional Information Author Contributions",
"section_num": null
},
{
"section_content": "All authors have reviewed the final version to be published and agreed to be accountable for all aspects of the work. ",
"section_name": "Additional Information Author Contributions",
"section_num": null
},
{
"section_content": "Acquisition, analysis, or interpretation of data: Koichiro Shinji, Tai-ichiro Chikama, Atsuhiko Fukuto, Yoshiaki Kiuchi, Taka-aki Moriguchi ",
"section_name": "Concept and design: Koichiro Shinji",
"section_num": null
},
{
"section_content": "Critical review of the manuscript for important intellectual content: Tai-ichiro Chikama, Atsuhiko Fukuto, Yoshiaki Kiuchi, Taka-aki Moriguchi Supervision: Tai-ichiro Chikama, Yoshiaki Kiuchi ",
"section_name": "Drafting of the manuscript: Koichiro Shinji",
"section_num": null
},
{
"section_content": "Human subjects: Consent was obtained or waived by all participants in this study.the Institutional Review Board of Hiroshima University issued approval E-709.Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work.Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work.Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. ",
"section_name": "Disclosures",
"section_num": null
}
] |
10.1038/s41408-019-0220-x
|
Association of elevated serumfree light chains with chronic lymphocytic leukemia and monoclonal B-cell lymphocytosis
|
<jats:title>Abstract</jats:title><jats:p>Chronic lymphocytic leukemia (CLL) and its precursor, monoclonal B-cell lymphocytosis (MBL), are heritable. Serumfree light-chain (sFLC) measures are a prognostic factor for CLL, but their role in susceptibility to CLL is not clear. We investigated differences between sFLC measurements in pre-treatment serum from five groups to inform the association of sFLC with familial and sporadic CLL: (1) familial CLL (<jats:italic>n</jats:italic> = 154), (2) sporadic CLL (<jats:italic>n</jats:italic> = 302), (3) familial MBL (<jats:italic>n</jats:italic> = 87), (4) unaffected first-degree relatives from CLL/MBL families (<jats:italic>n</jats:italic> = 263), and (5) reference population (<jats:italic>n</jats:italic> = 15,396). The percent of individuals having elevated monoclonal and polyclonal sFLCs was compared using age-stratified and age- and sex-adjusted logistic regression models. In age groups >50 years, monoclonal sFLC elevations were increased in sporadic and familial CLL cases compared to the reference population (<jats:italic>p</jats:italic>’s < 0.05). However, there were no statistically significant differences in sFLC monoclonal or polyclonal elevations between familial and sporadic CLL cases (<jats:italic>p</jats:italic>’s > 0.05). Unaffected relatives and MBL cases from CLL/MBL families, ages >60 years, showed elevated monoclonal sFLC, compared to the reference population (<jats:italic>p</jats:italic>’s < 0.05). This is the first study to demonstrate monoclonal sFLC elevations in CLL cases compared to controls. Monoclonal sFLC levels may provide additional risk information in relatives of CLL probands.</jats:p>
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) has an underlying heritable predisposition, with ~10% of individuals affected with CLL reporting a first-degree relative with CLL or a related lymphoproliferative disorder [1][2][3] .Monoclonal Bcell lymphocytosis (MBL), precursor disease to CLL, is an asymptomatic hematologic condition characterized by small absolute levels of blood B-cell clone and no other signs of a lymphoproliferative disorder [3][4][5][6][7] .In the general population, MBL increases with age with a prevalence of 5-9% in individuals over age 60 years 7 .We have reported MBL to be higher among first-degree relatives from CLL families, occurring at a higher rate in high-risk CLL families, implying a shared inherited risk 4 . Paraproteins, usually aligned with plasma cell disorders, have also been found to be prevalent in other B-cell malignancies, such as CLL 8 .In 2007, Martin et al. 9 evaluated the frequency of monoclonal serum-free light-chain (sFLC) measurements in patients with other B-cell malignancies and established abnormal sFLCs can be detected in a substantial proportion of patients with Non-Hodgkin's lymphoma (NHL) and CLL, and may be a useful clinical tool in the early diagnosis of a B-cell malignancy.Since then other studies have shown that sFLCs and the associated κ/λ ratio (rFLC) can also be used to inform prognosis, survival, and clinical outcomes in patients with CLL [10][11][12][13][14][15][16][17] .Maurer et al. 13 found that both monoclonal and polyclonal sFLC elevations were associated with poor overall survival in CLL compared to patients with normal sFLCs, even after adjusting for Rai stage.A prospective study showed an abnormal rFLC can be detected several years before the actual diagnosis of CLL in a significant percentage of patients 18 .Thus, sFLC measurements have been implicated in detection and prognosis of CLL, however, few studies evaluating sFLCs and risk of CLL (or MBL) have been performed. Further, there is limited evidence on the differences between sporadic and familial CLL, with most studies focusing on prognostic and clinical factors.Stage at initial diagnosis, need for treatment, and survival over a 10-year period have been reported to be similar in sporadic and familial cases 19 .Similarly, no differences in the expression of ZAP-70, CD38, and CD23, and levels of serum β2microglobulin have been reported 20 .Studies that examined the mutational status of genes in the immunoglobulin heavy chain variable region (IgVH), known to be associated with a better prognosis, found higher frequency of mutated CLL in familial compared to sporadic CLL 21 .Another study found differences in frequency of VH gene segments (4-34, 5-51, 1-69, 4-29, 3-23) in familial vs. sporadic disease 22 .Among familial cases, VH gene segment utilization proved non-random and diverged from the frequencies previously reported among unrelated patients with CLL 22 .However, the association of sFLC with familial and sporadic CLL has not been performed. In this study, for the first time, we investigated the association of sFLC levels with risk of CLL (or MBL) using CLL cases (familial and sporadic), unaffected relatives, and a large comparable reference population.We hypothesized that pre-treatment sFLC elevation would be higher among familial vs. sporadic CLL, and in CLL cases compared to the reference population.Second, given the known familial nature of CLL, we hypothesized that pretreatment sFLC would be elevated in the family members of patients with CLL (both unaffected relatives and those with MBL) compared to the reference population. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "All participants provided written informed consent.The protocol was approved by the Institutional Review Board at each participating center.Individuals with pretreatment serum samples were eligible.Those known to have died within 1 year of sample date were excluded due to likelihood of underlying disease, which could falsely inflate sFLC levels.Five participant groups (primarily Caucasian) were defined for analyses consisting of three case groups and two control groups: (1) Sporadic CLL cases, (2) Familial CLL cases, (3) Familial MBL cases from CLL / MBL families, (4) Unaffected relatives from CLL/ MBL families; and (5) a reference population (Olmsted County controls) [23][24][25][26] . ",
"section_name": "Study participants",
"section_num": null
},
{
"section_content": "Sporadic CLL cases consisted of incident CLL cases diagnosed from 2002-2008 without a self-reported family history of CLL and/or MBL and with a pre-treatment sFLC measure in the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER) 13 . ",
"section_name": "Sporadic CLL cases",
"section_num": null
},
{
"section_content": "Familial CLL cases were selected from families with at least two reported CLL and/or MBL cases.Families were recruited through the Genetic Epidemiology of CLL (GEC) consortium 27 ",
"section_name": "Familial CLL cases",
"section_num": null
},
{
"section_content": "In our GEC families described above, relatives were screened for MBL in accordance with our previous work 4 .MBL status was determined by flow cytometry and was defined at the time of screening.Any blood relative found to have MBL in a family with two or more CLL and/or MBL cases was eligible for inclusion.We did not require a specific degree of relation between cases. ",
"section_name": "Familial MBL cases",
"section_num": null
},
{
"section_content": "Family members with no known CLL, MBL, or any other lymphoproliferative disorder who were a firstdegree relative to at least one CLL or MBL case from a familial CLL family were considered unaffected relatives.Primarily, MBL screening was performed on a baseline sample, which was also used for the sFLC measurements; however, there were some samples that did not get screened and were assumed to be unaffected. ",
"section_name": "Unaffected relatives from CLL/MBL families",
"section_num": null
},
{
"section_content": "The reference population was a previously characterized population-based cohort of Olmsted County residents age 50 years or older 23 .From 1 January, 1995, to 21 November, 2003, serum samples were obtained from 21,462 (76.6%) of 28,038 enumerated Olmsted County residents and tested for monoclonal protein levels and FLC.We refer to this group as \"controls\" as they have no known diagnosed CLL or precursor disease, MBL. Of the 21,462 controls, we excluded 577 individuals with known plasma cell disorders, including those with monoclonal gammopathy of undetermined significance (MGUS), because these are the disorders in which clonal elevations of FLC are known to occur 28 .However, we did not exclude light chain (LC)-MGUS, since FLC is used in the definition of this condition.We also excluded 4,096 individuals who declined authorization to participate in research 29 and 758 who did not have sufficient sample volume to perform the FLC assay.Of the remaining 16,031 subjects, we also excluded individuals with a known lymphoproliferative disorder or precursor (n = 62) or a first-degree relative with this condition (n = 32) from clinical or research databases.After excluding individuals whose death date was within 1 year of sample date (prevalent disease: n = 541), the final control group consisted of 15,396 subjects. ",
"section_name": "Reference population: Olmsted County (controls)",
"section_num": null
},
{
"section_content": "Heparinized plasma or serum samples were tested using the sFLC assay (FREELITE™, The Binding Site Ltd., Birmingham, UK) to measure κ and λ immunoglobulin FLC levels at either the Mayo Clinic or National Cancer Institute (NCI) 23 .Prior studies have shown the high intraclass correlation between FLC measures using fresh and stored serum and for the FREELITE™ kappa and lambda free light chains assay; both EDTA-plasma and lithium heparin-plasma can serve as acceptable substitutes for serum 30,31 .As previously described, the analytic sensitivity for the nephelometric FLC assay is 0.1 mg/ L for a monoclonal κ or λ FLC 32 .For samples analyzed at both NCI and Mayo Clinic, elevated FLC was defined as a κ or λ level above the reference range (κ > 19.4 mg/L, λ > 26.3 mg/L) 13,33 .Likewise, an elevated FLC was considered monoclonal if the rFLC (κ/λ) was outside of the normal range of 0.26-1.65 13,33 .Elevated FLC levels with normal rFLC (κ/λ) were considered as polyclonal.Patients with normal FLC levels but an abnormal rFLC (κ/λ) were considered to have ratio-only FLC abnormalities and not considered in the evaluation of elevated FLC 13 .We also evaluated the absolute value or sum of the free light chains, which has utility in non-clonal disease situations and has been shown to predict survival in the general population 23 .The timing of the plasma or serum samples was a median of 2 months between diagnosis date and blood draw date for sporadic CLL cases (N = 302) (range = (0 years-5.7 years), IQR = (2 weeks-9 months)), and a median 18 months between diagnosis and blood draw date for familial CLL cases (N = 154) (range = (0 years-25 years), IQR = (6 months-4.2years)).Additionally, the FLC was measured in the baseline sample for the majority of subjects, therefore corresponding baseline MBL status was used.Some of the subjects may have had MBL identified in a follow-up sample, but for this study we use the MBL status at the time of the FLC measurement. ",
"section_name": "Free lightchain testing",
"section_num": null
},
{
"section_content": "Analyses of FLC levels were conducted between the following groups: (1) Sporadic CLL cases vs. familial CLL cases; (2) Sporadic CLL cases vs. controls; (3) Familial CLL cases vs. controls; (4) Familial MBL vs. controls, and (5) Unaffected relatives in CLL/MBL families vs. controls.Age at FLC was summarized as mean ± standard error (SE), and two-sample t-tests were used for age comparisons across groups.Sex and percent elevated FLC were summarized by group as counts and percentages, respectively.The percent of individuals having elevated FLC was compared using age-and sex-adjusted logistic regression models.The FIRTH option 34 was applied to logistic regression to test for significance when there were small numbers in particular cells and estimates were thought to be unstable.Multivariable linear regression models were used to calculate the least squares (LS) means of κ, λ, κ/λ ratios, and FLC sum after adjusting for age and sex; due to the non-normal distributions of the FLC measures, we first log-transformed the values, performed the calculations, then exponentiated the values to summarize. We performed secondary analyses to ensure that statistical differences were not due to relatedness.We randomly chose one relative from each family from each relative group (either one unaffected or one MBL) and reran analyses above. Due to the age of the reference population, analyses that compared to the reference group were limited to individuals who were age 50 years and older.Comparisons between sporadic and familial CLL cases included cases who were age 35 and older at sample collection; sensitivity analyses were performed subsetting to age 50 years. Analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC). ",
"section_name": "Statistical analyses and considerations",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "We identified 302 sporadic CLL cases from MER, 154 familial CLL cases, 87 familial MBL cases, 263 unaffected first-degree relatives from GEC, and 15,396 subjects from the Olmsted County reference population, otherwise referred to as controls. ",
"section_name": "Study participants",
"section_num": null
},
{
"section_content": "Characteristics and comparisons between sporadic CLL and familial CLL cases are shown in Table 1.Sporadic and familial cases were similar in age, however, sporadic cases were comprised of more males (68%) compared to familial cases (49%) (Table 1).The adjusted mean FLC sum (familial = 2.75, sporadic = 2.70; p-value = 0.73) and rFLC (familial = 1.03, sporadic = 1.13; p-value = 0.42) were similar in both CLL case groups.When investigating the percent elevated FLC (monoclonal and polyclonal combined) by age group, there was no statistically significant difference between familial and sporadic CLL cases.Likewise, when examining specific type of elevation, there were no statistically significant differences between the familial and sporadic cases (p's > 0.05). ",
"section_name": "Comparison of sporadic CLL and familial CLL cases",
"section_num": null
},
{
"section_content": "Characteristics and comparisons between CLL cases (familial and sporadic) and controls are shown in Table 1.The mean age of familial CLL cases (64.2 years) was higher than controls (63.9 years), while the mean age of sporadic CLL cases (63.2 years) was slightly lower (p < 0.003).There were higher proportions of females in the familial cases (51%) and control groups (55%) compared to sporadic cases (32%) (Table 1).Mean lambda FLC values (λ-FLC; mg/L) were significantly lower in familial (1.19 mg/L) and sporadic (1.13 mg/L) CLL cases compared to the controls (1.51 mg/L) (p < 0.001).Mean kappa FLC values (κ -FLC; mg/L) were similar across groups: 1.22 mg/L for familial cases, 1.27 mg/L for sporadic cases, and 1.24 mg/L for controls (p > 0.05).The rFLC (κ:λ) was significantly higher in familial (1.03) and sporadic (1.13) CLL cases compared to the controls (0.82) (p's < 0.001).Percent elevated FLC (polyclonal and monoclonal combined) was significantly higher in both familial and sporadic CLL cases compared to the controls in younger age groups (p < 0.05).Specifically, in ages 50-59 years, percent elevated FLC was ~12% higher in familial (22%) and sporadic (21%) cases compared to controls (9%) (p < 0.01).In ages 60-69 years, percent elevated FLC was ~11% higher in cases compared to controls (16%); this was seen in both sporadic (28%; p = 0.009) and familial cases (28%, p = 0.06).In ages 70-79 years, percent elevated FLC was significantly higher (~21%) in sporadic cases (47%) compared to controls (26%; p = 0.01).Although not statistically significant, familial cases (38%) also had a higher percent elevated FLC (~12%) compared to controls (26%) (p = 0.18).However, at older ages (80 + years), we did not see a statistically significant difference between percent elevated FLC in sporadic (44%) or familial (59%) cases compared to controls (42%) (p = 0.89 and p = 0.18, respectively). When separating elevated FLC into monoclonal and polyclonal elevation, there was no statistically significant difference between percent of polyclonal FLC in sporadic (7%, 17%, 22%) or familial (2%, 12%, 19%) cases compared to controls (9%, 15%, 25%), for age groups 50-59, 60-69, and 70-79, respectively (p's > 0.05).In the oldest age group (80 + years), sporadic CLL cases had lower percent polyclonal FLC (11%) compared to controls (41%; p = 0.01), however, the sample size in this group was small (n = 2/24).However, a statistically significant (p < 0.001) increase in monoclonal FLC elevation was observed in both familial (20%, 16%, 19%, 24%) and sporadic (13%, 11%, 25%, 33%) CLL cases compared to the controls (0.5%, 0.7%, 1%, 1%) across age groups 50-59, 60-69, 70-79, and 80 + , respectively.In a sensitivity analysis of familial and sporadic cases (ages 50 + years at sample collection), results remained similar (Supplementary Table 1). ",
"section_name": "Comparison of CLL cases (sporadic/familial) and reference population (controls)",
"section_num": null
},
{
"section_content": "Characteristics and comparisons between familial MBL, unaffected relatives from CLL/MBL families, and controls are shown in Table 2. Relatives with MBL were on average older (68.4 years) than unaffected family members (63.6 years) or controls (63.9 years); however, the difference was only statistically significant for familial MBL compared to controls (p < 0.001).The proportion of females was significantly higher among the unaffected relatives (68%) compared to the controls (55%; p < 0.001).Familial MBL cases and unaffected relatives both had a higher age and sex-adjusted mean serum rFLC than the controls, although values were all within the normal range (0.26 to 1.65) 13,33 , (rFLC = 1.04 for familial MBL, 1.00 for unaffected family members, and 0.82 for controls, respectively (p < 0.001)).Similar trends were observed when the analyses were repeated using one randomly selected relative from each family per category (Supplementary Table 2). When investigating the percent elevated FLC by age group (50-59, 60-69, 70-79, and 80+), there were no statistically significant differences between familial MBL (8%, 14%, 31%, 50%) or unaffected relatives (5%, 14%, 20%, 50%) compared to controls (9%, 16%, 26%, 42%).However, when looking at types of FLC elevation, there were statistically significant differences in monoclonal, but not polyclonal, elevation in family members in most of the age groups 60 and older, including MBL cases (5%, 7%, 8%) and unaffected relatives in CLL / MBL families (3%, 2%, 8%), compared to the Olmsted County controls (2%, 1%, 1%) for ages 60-69, 70-79 and 80+, respectively (all p's < 0.05); the only exception was the age 70-79 group of unaffected relatives compared to controls (p = 0.25).Analyses restricted to only one family member showed generally similar results, although statistical significance varied by age group (Supplementary Table 2). ",
"section_name": "Comparisons of familial MBL, unaffected relatives, and reference population (controls)",
"section_num": null
},
{
"section_content": "We demonstrate increased monoclonal elevations for CLL cases, regardless of family history, compared to a reference population.Monoclonal elevation in both sporadic and familial CLL cases was higher than in the reference population.We also found evidence for increased monoclonal elevation in relatives, including unaffected family members as well as those with MBL, compared to that of the reference population, which may provide additional risk information in unaffected and MBL relatives of CLL probands. We report no differences in sFLC elevation between familial and sporadic CLL cases, which is consistent with prior reports that have demonstrated little or no difference in other clinical and prognostic markers between these two groups 19,20,[35][36][37][38] .For example, no differences in the expression of biologic markers (ZAP-70, CD38, and CD23), levels of serum β2-microglobulin, CXCR4 expression, or chromosome 13q deletion have been reported 20,37,38 .Thus, our results from this study add to evidence for similar biology underlying sporadic and familial CLL. Our finding of an increased monoclonal, but not polyclonal, protein elevation among unaffected family members compared to the controls is provocative.Polyclonal FLC elevation, where the rFLC is normal, can be due to a number of causes including renal dysfunction, inflammation, or immune stimulation 39,40 , whereas a monoclonal elevation is suggestive of clonal plasma cell proliferation (e.g., multiple myeloma/MGUS) 41,42 .In a prior study of CLL, the monoclonal elevated light chain in the serum (kappa or lambda) agreed with the clonal Bcell's lightchain restriction by flow cytometry in 96% of cases 13,18 .In addition to elevated sFLC measures in our Our study has a number of strengths, including a wellcharacterized collection of familial and sporadic CLL cases, all of whom had validated CLL diagnoses through medical record review, and a large comparable and systematically screened population-based reference group.Limitations include the small sample size for some of the age groups examined and generalizability to other ethnicities due to the primarily Caucasian nature of our families and Olmsted County reference population (controls).Additionally, we make the assumption that the Olmsted County population is unaffected, as they are not screened for MBL, however to the best of our knowledge they have no known reported CLL.Regarding the unaffected relatives from the GEC study sites, primarily the same baseline sample was used for the sFLC measurements and the MBL screening, however, there were some samples that did not get screened for MBL and were assumed to be unaffected.Finally, we did not have total serum protein or the flow cytometry results available to correlate the lightchain restriction pattern with the sFLC results.However, prior literature suggests high concordance between monoclonal elevated light chain in serum and the CLL clone's lightchain restriction 13,18 .Another important consideration is we were unable to perform analyses including other prognostic parameters due to missingness for familial CLL cases.Future studies should aim to consider prognostic parameters (e.g., beta-2-microglobulin, Rai Stage, IgHV mutational status, FISH cytogenetics, CLL-IPI index) with sFLC in sporadic vs. familial context. Our study showed that sporadic and familial CLL have similar sFLC but increased monoclonal sFLC elevation compared to controls.In addition, monoclonal elevation of sFLC was higher among unaffected relatives and relatives with MBL from CLL families than a general population.To our knowledge, this is one of the first studies to examine sFLC among relatives from CLL/MBL families and provide further evidence of the potential of monoclonal sFLC elevations as a valuable prognostic factor in CLL/MBL.Follow-up studies are needed to replicate our findings and determine the relationship between elevated sFLC levels, MBL, and future CLL risk. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "This work was supported by National Institutes of Health grants Mayo Clinic Lymphoma SPORE: P50 CA97274; U01 CA118444 (CLL/Slager), R01 CA92153 (CLL/Cerhan), R25 CA092049, R01 CA134674 (CLL/Camp).This study was funded by Mayo Clinic Lymphoma SPORE: P50 CA97274; U01 CA118444 (CLL/ Slager), R01 CA92153 (CLL/Cerhan), R25 CA092049, R01 CA134674 (CLL/Camp). ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Author details 1 Division of Epidemiology, Department of Health Sciences, Mayo Clinic, Rochester, MN, USA. 2 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/s41408-019-0220-x). ",
"section_name": "Conflict of interest",
"section_num": null
},
{
"section_content": "Author details 1 Division of Epidemiology, Department of Health Sciences, Mayo Clinic, Rochester, MN, USA. 2 ",
"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/s41408-019-0220-x). ",
"section_name": "Conflict of interest",
"section_num": null
}
] |
10.3390/cancers14153604
|
Update in Pathogenesis, Diagnosis, and Therapy of Prolactinoma
|
<jats:p>Prolactinomas comprise 30–50% of all pituitary neuroendocrine tumors, frequently occur in females aged 20 to 50, and cause hypogonadism and infertility. In typical cases, female patients exhibit galactorrhea and amenorrhea due to serum prolactin (PRL) elevation, and patients during pregnancy should be carefully treated. During diagnosis, other causes of hyperprolactinemia must be excluded, and an MRI is useful for detecting pituitary neuroendocrine tumors. For treating prolactinoma, dopamine agonists (DAs) are effective for decreasing PRL levels and shrinking tumor size in most patients. Some DA-resistant cases and the molecular mechanisms of resistance to a DA are partially clarified. The side effects of a DA include cardiac valve alterations and impulse control disorders. Although surgical therapies are invasive, recent analysis shows that long-term remission rates are higher than from medical therapies. The treatments for giant or malignant prolactinomas are challenging, and the combination of medication, surgery, and radiation therapy should be considered. Regarding pathogenesis, somatic SF3B1 mutations were recently identified even though molecular mechanisms in most cases of prolactinoma have not been elucidated. To understand the pathogenesis of prolactinomas, the development of new therapeutic approaches for treatment-resistant patients is expected. This review updates the recent advances in understanding the pathogenesis, diagnosis, and therapy of prolactinoma.</jats:p>
|
[
{
"section_content": "Prolactinomas are common pituitary neuroendocrine tumors (PitNET) derived from prolactin (PRL)-producing cells that cause hypogonadism and infertility due to hyperprolactinemia.It most frequently occurs in young women and is often associated with galactorrhea and amenorrhea.Most cases are sporadic, but prolactinomas may also occur because of mutations of the multiple endocrine neoplasia type 1 (MEN1) or aryl hydrocarbon receptor-interacting protein (AIP) gene.Medical therapy with a dopamine agonist (DA) is highly effective in the majority of cases [1,2]. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "Prolactinoma accounts for approximately 50% of all pituitary tumors and is the most frequent of all functional PitNETs [2,3].The annual incidence of prolactinomas is about 2.2 per 100,000 in Finland [4].It is most common in women of reproductive age, with an incidence of about 10 per 100,000.Peak incidence occurs around 30 years old [5] but is also seen after menopause.The annual incidence of DA-treated hyperprolactinemia is 24 per 100,000 women aged 25 to 34 in the Netherlands [6].Male patients are relatively rare, and the disease takes longer to be diagnosed because of the lack of clinical symptoms. ",
"section_name": "Epidemiology",
"section_num": "2."
},
{
"section_content": "Prolactinomas are divided into two subgroups: familial and sporadic.Among the familial cases, the well-known causative mutation is that of MEN1.Indeed, prolactinoma is most frequent in familial PitNETs in patients with MEN1 [7].Although relatively rare, MEN1 gene mutation is found in sporadic prolactinomas.In addition, prolactinoma is reported to occur in families with a mutation in the PRKAR1A (Carney complex), CDKN1B (MEN4), or AIP (FIPA) gene [8]. The precise molecular mechanism whereby MEN1 gene mutation causes prolactinoma is still obscure.In endocrine cells, MENIN, a protein product of the MEN1 gene, has a negative effect on cell growth via the induction of cell cycle-inhibiting genes such as CDKN1B (p27 KIP1 ) or CDKN2C (p18 IKN4C ).Thus, MEN1 is recognized as a tumor-suppressor gene.In this sense, a loss-of-function mutation in one allele of genomic DNA can enhance the cell cycle, followed by the tumorous growth of the affected cells.The reason that the germ line MEN1 gene mutation causes a PRL-, growth hormone (GH)-or thyroid stimulating hormone (TSH)-producing PitNET as opposed to any other pituitary tumor is not known.One possibility is that MENIN somehow interacts with PIT1 (POU1F1), the common transcription factor specifically expressed in the somato-, mammo-, and thyrotrope cells, and facilitates PIT1-dependent gene transcription that is related to hormone synthesis and cell growth. The PRKAR1A gene encodes the regulatory subunit of protein kinase A (PKA), and a germ line loss-of-function mutation can cause constitutive activation of the catalytic subunit of PKA with a resultant increase in PKA-dependent cell growth and the production of pituitary hormones such as PRL, GH, or TSH.However, the impact of PKA activation on cell proliferation is moderate, and the affected cells are reported to show the phenotype of non-invasive hypertrophy rather than an aggressive tumor. Regarding sporadic pituitary tumors, recent advances in genome-wide sequencing (GWAS) have identified the mutations responsible for tumorigenesis.Indeed, USP8 and gsp gene mutations have been found in the substantial parts of corticotrope and somatotroph tumors, respectively.Until recently, however, no driver-gene mutation had been reported for prolactinoma, partly because most patients with the tumor receive medical, not surgical, therapy.Recently, a GWAS analysis of somatotroph tumor cells showed aberrant splicing of various mRNAs, including that of the estrogen-related receptor γ (ERRγ) gene (ESRRG) [9].Eventually, Li et al. identified a missense mutation of the splicing factor 3B1 gene (SF3B1), causing the amino acid substitution (R625H) [10].Because the SF3B1 protein is involved in splicing nascent RNA, a variety of abnormally spliced mRNA can result from this mutation.In the case of lactotroph tumor cells, abnormally spliced mRNA derived from the ESRRG gene, which encodes ERRγ, results in a mutant ERRγ protein, which has shown to have an abnormally high affinity for PIT1 and potently enhance PIT1-dependent PRL gene transcription.Furthermore, the expression of the DLG1 protein is found to be decreased due to the aberrantly spliced DLG1 mRNA [11].The DLG1 protein is known to have tumor-suppressor properties, and thus its decrease can cause enhanced lactotroph cell growth.Indeed, a prolactinoma-harboring SF3B1 gene mutation is reported to have invasive properties. The pathogenesis of prolactinoma without the SF3B1 mutation is still unknown.One of the major factors promoting lactotroph tumorigenesis is estrogen; indeed, prolactinoma is known to be more frequent in women than in men.In an animal experiment, prolactinoma was reported to develop a lactotroph tumor in some species of rats in which estrogen was chronically administered.Estrogen is known to increase the expression of a variety of growth factors and oncoproteins such as fibroblast growth factors (FGFs), transforming growth factor β (TGF β), and pituitary tumor transforming gene (PTTG), all of which are known to facilitate tumor growth.More recently, it was reported that estrogen causes epigenetic changes in a variety of tissues other than the adenohypophysis [12].If estrogen changes the expression level of genes such as the dopamine D2 receptor via epigenetic changes to the pituitary cells, the hormone may influence lactotroph cell growth without a gene mutation.The involvement of an epigenetic mechanism in prolactinoma pathogenesis is not well characterized and awaits further research. ",
"section_name": "Pathogenesis",
"section_num": "3."
},
{
"section_content": "PRL is secreted from the lactotroph in the anterior pituitary gland.PRL is regulated mainly by hypothalamic dopamine in an inhibitory manner and by thyrotropin-releasing hormone (TRH) and estrogen in a stimulatory manner.Physiologically, PRL has a positive effect on mammary gland development during pregnancy and milk secretion during the postpartum period.Eating, exercise, and sleep increase serum PRL levels, and they are influenced by the menstrual cycle, pregnancy, and female hormone therapy.These factors should be clarified during a medical interview [1,2,13]. Hyperprolactinemia causes symptoms such as galactorrhea and amenorrhea, the former being due to the direct effect of PRL on the mammary glands and the latter due to the suppression of hypothalamic gonadotropin-releasing hormone (GnRH) and pituitary gonadotropin secretion.It is known that hyperprolactinemia inhibits GnRH neurons via kisspeptin neurons [14].In men, the suppression of the gonadal system also elicits a decreased libido and pubescence, which are often unrecognized.As a result, headache and visual field disturbance associated with macrotumors are frequently the main complaints in men [1,2,13].In addition, hypogonadism is a common form of hypopituitarism in men with macroprolactinoma, and the entity of hypogonadism recovery after treatment is a relevant medical issue, mainly in younger patients [15]. Serum PRL levels are in a supraphysiological range during pregnancy and the postpartum period.Since basal serum PRL levels are prone to fluctuation, especially in women, multiple measurements should be performed to ensure reproducibility.Known causes of hyperprolactinemia include prolactinoma, acromegaly (growth-hormone-producing PitNET), hypothalamic and pituitary stalk lesions, medications, primary hypothyroidism, and renal failure [16] (Table 1).In cases of severe hyperprolactinemia (>200 ng/mL), the presence of a prolactinoma should be considered, although it can present with any level of PRL elevation [3,17].In mild PRL elevation, drug-induced hyperprolactinemia should be considered.The causes of drug-induced hyperprolactinemia include anti-ulcer and antiemetic drugs, antihypertensive drugs, psychotropic drugs, and estrogens.To determine whether it is drug-induced or not, the candidate drug could be discontinued or replaced by other drugs to see if PRL levels return to a normal range within 48-96 h [18].Almost half of GH-producing PitNETs (acromegaly) simultaneously secrete PRL.An organic lesion in the hypothalamus and pituitary stalk, such as tumors, inflammation, and granulomatous and vascular diseases, could cause hyperprolactinemia, and thus, it is necessary to exclude these diseases.As mentioned above, inhibitory regulation by hypothalamic dopamine is the predominant regulatory mechanism of PRL secretion.Mass lesions of the hypothalamus or pituitary stalk cause the dopamine inhibition of PRL to be insufficient, resulting in hyperprolactinemia.In primary hypothyroidism, the release of a negative feedback regulation can lead to increased hypothalamic TRH secretion, resulting in hyperprolactinemia.If Cancers 2022, 14, 3604 4 of 13 no clinical symptoms are observed in patients with hyperprolactinemia, the possibility of macroprolactinemia should be considered.Macroprolactin consists of high molecular weight PRL, which is a complex of PRL and anti-PRL antibodies and exhibits markedly reduced biological efficacies.In this case, serum PRL levels should be re-assayed after anti-PRL antibodies have been removed by polyethylene glycol treatment [19].If underlying causative diseases cannot be found, idiopathic hyperprolactinemia is diagnosed.Serum PRL levels were stable in most idiopathic cases after long-term follow up [20,21]. Prolactinoma is diagnosed by imaging tests, such as magnetic resonance imaging (MRI), which detects PitNETs.Additionally, hyperprolactinemia can be caused by a variety of other factors, as described above, and thus these have to be excluded.Serum PRL should be measured in suspected cases of prolactinoma: women with irregular menstruation, amenorrhea, infertility, or galactorrhea, and men with decreased libido.After hyperprolactinemia is confirmed, various differential diagnoses should be made, including prolactinoma, medications that cause hyperprolactinemia, or organic diseases in the hypothalamic/pituitary region.Of these, drug-induced hyperprolactinemia is the most common.When prolactinoma is suspected, MRI imaging is warranted.Although microtumors are not usually obvious on simple MRI scans, gadolinium contrast-enhanced MRI can clearly detect them in most cases (Figure 1).In prolactinomas, tumor volume usually correlates with serum PRL levels.In the case of giant prolactinoma and moderately elevated PRL levels, the hook effect should be suspected.The hook effect is due to saturation of the assay antibodies, and diluted samples should be used for PRL measurement [22].Regarding an endocrinological examination, the TRH loading test in prolactinomas often shows high basal PRL with low response.However, it is not specific and should be used as an ancillary test.The bromocriptine loading test is helpful for predicting the effectiveness of DA, as it suppresses PRL to more than half the basal value [1]. Cancers 2022, 14, x 5 of 13 (3) Ectopic prolactin-producing tumor (4) Idiopathic hyperprolactinemia The table was created by modifying The Guide to Diagnosis and Treatment of Hypothalamic Pituitary Disease [23]. ",
"section_name": "Diagnosis",
"section_num": "4."
},
{
"section_content": "The goals of prolactinoma treatment are to counteract hypogonadism by suppressing hyperprolactinemia and decreasing the size of the tumor [1,2].Pharmacotherapy is the first-line treatment for prolactinomas, and dopamine agonists (DAs) represent the primary therapy for almost all prolactinomas, including microtumors (less than 1 cm in diameter, macrotumors (greater than 1 cm), or giant tumors (greater than 4 cm).DAs, such as bromocriptine or cabergoline, are highly effective in suppressing PRL secretion and reducing tumor size (Figure 1).Long-acting DA (cabergoline) is often used in clinical prac- ",
"section_name": "Medical Treatment",
"section_num": "5."
},
{
"section_content": "The goals of prolactinoma treatment are to counteract hypogonadism by suppressing hyperprolactinemia and decreasing the size of the tumor [1,2].Pharmacotherapy is the first-line treatment for prolactinomas, and dopamine agonists (DAs) represent the primary therapy for almost all prolactinomas, including microtumors (less than 1 cm in diameter, macrotumors (greater than 1 cm), or giant tumors (greater than 4 cm).DAs, such as bromocriptine or cabergoline, are highly effective in suppressing PRL secretion and reducing tumor size (Figure 1).Long-acting DA (cabergoline) is often used in clinical practice because it is effective in small intermittent doses (0.25-0.5 mg once a week) without major side effects.Dosage reduction or discontinuation should be considered when normalization of serum PRL levels and disappearance of pituitary tumors are achieved after two or more years of DA treatment [1,24]. The normalization rate of serum PRL with bromocriptine was 78 and 72% for patients with microtumor and macrotumor, respectively [24].Cabergoline is highly effective in normalizing serum PRL levels and reducing tumor size.In female patients treated with cabergoline, 83% achieved a normalized PRL; 72% had a recovered menstrual cycle, and only 3% discontinued because of adverse effects.Of those treated with bromocriptine, 59% achieved a normalized PRL; 52% had a recovered menstrual cycle, and 12% discontinued because of adverse effects [25].In another study, cabergoline normalized PRL levels in 92% of patients with microprolactinoma and in 77% of patients with macroprolactinoma [26].Cystic prolactinomas were thought to be resistant to drug therapy, but recently cabergoline has been shown to be beneficial [27].Drug therapy for giant prolactinomas should be continued if they respond well.If the response to DAs is insufficient, surgical treatment should be considered to debulk the tumor volume, which may improve postoperative medical control. DAs may produce side effects such as gastrointestinal symptoms, orthostatic hypotension, and nasal obstruction, as well as psychiatric symptoms, such as depression, anxiety, and insomnia [1,2].The intravaginal administration of bromocriptine was effective in women and had diminished gastrointestinal side effects [28].Furthermore, impulse disorders (e.g., pathological gambling, hypersexuality, and compulsive shopping or eating) Cancers 2022, 14, 3604 6 of 13 have been described.The mechanism of impulse disorder is considered as DAs effect on the mesolimbic system via the subtype 3 dopamine receptor (D3 receptor).Discontinuing DAs usually reverses these side effects [29,30].Long-term treatment with DAs may cause valvular heart disease.Although a large follow-up study did not support a clinical connection between DAs treatment and cardiac valvular disease, a meta-analysis that evaluated patients who received cabergoline treatment reported an increased risk of tricuspid regurgitation [31,32].An echocardiogram should be conducted for patients with an audible murmur, those treated for more than 5 years with a dose of more than 3 mg per week, or those who maintained cabergoline treatment after age 50 [33].In addition, when DAs are used for giant tumors invading the skull base, spinal fluid rhinorrhea and meningitis may occur as the tumor shrinks.In this case, patients are treated with antibiotics, or if necessary, the skull base is repaired by neurosurgery.Discontinuing DAs is not recommended because it may cause tumor relapse [34,35]. Prolactinomas are sometimes resistant to DA pharmacotherapy, and the estimated prevalence of DA resistance is 20-30% for bromocriptine and around 10% for cabergoline.In a study of 122 patients with macroprolactinoma, 80% of bromocriptine-resistant patients achieved normal PRL levels using cabergoline, and most patients resistant to standard doses of cabergoline responded to larger doses [24,[36][37][38].DA resistance was more prevalent in macroprolactinomas, invasive tumors, and male patients; 79% achieved normal PRL levels with a standard dose of cabergoline; 15% required higher doses; 6% were resistant.A T2-weighted MRI intensity of prolactinoma may help predict the response to DAs: the heterogeneity of tumoral T2 signal could be used as a predictive factor of DA resistance [39].Regarding molecular mechanisms, DA resistance is associated with reduced subtype 2 dopamine receptor (D2 receptor) expression, particularly in the long-acting form of the D2 receptor [40,41].In addition, factors downstream of the D2 receptor might contribute to DA inhibition, such as alterations in cytoskeleton protein filamin A or nerve growth factor (NGF) receptors [42,43].Transforming growth factor beta (TGFβ) is also identified as a potential molecule for inducing DA resistance [44,45]. ",
"section_name": "Medical Treatment",
"section_num": "5."
},
{
"section_content": "The common indications for surgery in prolactinomas are resistance or intolerance to DA or the failure of the maximum dose of DA to lower the PRL or reduce tumor volume in macroprolactinomas.Acute complications (e.g., pituitary apoplexy, CSF leakage, or symptomatic tumor enlargement during pregnancy in DA-resistant cases) are rare indicators.Debulking surgery may be considered for women with macroprolactinomas who are planning to become pregnant.Other recently seen indications are patients with mainly cystic tumors or younger patients unwilling to undergo long-term DA treatment whose tumor is likely to be completely resected [2]. DA resistance is defined as the inability to achieve a normalization of serum PRL levels and a 50% reduction in tumor size at least 3-6 months after the weekly administration of the maximum-tolerated dose of a DA: 15 mg of bromocriptine or 1.5-3.0mg of cabergoline [36].If side effects make it difficult to increase the dose of a DA, a dose lower than this is the maximum.However, side effects of cabergoline were recorded in 68% of women, but only a few patients discontinued it [46]. Most surgeries for prolactinomas are performed by transsphenoidal surgery, and endoscopic transsphenoidal surgery is currently the mainstream method.A craniotomy is required depending on the location of the tumor.In a meta-analysis, remission rates were similar in patients who underwent endoscopic and microscopic surgery.It was reported that the neurosurgeon's expertise, not the surgical approach, is more important for the surgical outcomes [2,47]. As for complications of transsphenoidal surgery, surgery-related mortality was 0%; persistent enuresis was 2%, meningitis was 1%, and the cerebrospinal fluid leak was 3%.In the meta-analysis, hypopituitarism was found in only 2%, hypoadrenocorticism in 1-2%, hypogonadism in 3-6%, and hypothyroidism in 1-6%.However, these risks are much greater in inexperienced centers [47].Since the main purpose of prolactinoma surgery is to restore gonadal function, it becomes meaningless if it results in central hypogonadism. A meta-analysis showed that the rate of gross tumor resection was inversely proportional to tumor size, with gross tumor resection achieved in 98-100% of microprolactinomas and 69-85% of macroprolactinomas.The overall postoperative and follow-up remission rates, having a median, mean follow-up period of 2.8 years using the random-effects model, were 0.62 and 0.61, respectively.The recurrence rate was 0.16 as assessed at a median follow-up period of 3.8 years [48].The recurrence rate after surgery increased as the follow-up period increased [49].However, in DA-resistant or intolerant patients, surgical tumor reduction may reduce, if not eliminate, the required DA dose or improve hormone control.Long-term remission has been reported to be highly controlled with a combination of surgery and a DA [46].Postoperative remission was more likely to occur in microprolactinomas than in macroprolactinomas, and giant prolactinomas (>4 cm) rarely went into remission from surgery alone [50].The success rate of surgery is highly dependent on the experience and skill of the neurosurgeon as well as the tumor size and degree of invasiveness.The remission rate was higher in centers with a large number of surgical cases than in centers with a small number [49].Preoperative visual disturbances and headaches improved in the majority of patients after surgery [51]. The presence of cavernous sinus invasion and the initial serum PRL levels have often been reported as predictors of a surgical outcome.Even in intracellular prolactinomas, the remission rate was higher for midline-located tumors than those that are laterally located [52].Similarly, prolactinomas with a lower Knosp grade have a higher remission rate [53].Preoperative PRL levels <200 ng/mL were associated with a higher rate of longterm remission [52].On the other hand, a preoperative PRL level >500 ng/mL is unlikely to result in a surgical cure, and >1000 ng/mL is unlikely to result in biochemical control [54]. A postoperative serum PRL level <10 ng/mL is associated with fewer recurrences [55,56]. In two meta-analyses of surgery versus DA, surgery resulted in 67-88% long-term remission after transsphenoidal surgery and 34-52% long-term remission after DA withdrawal, whereas the normalization of PRL with DA was achieved in 81% of cases [47,57].Another meta-analysis reported that although there was no difference in the three-month results between the surgical and medical treatment, the remission rate was higher in the medical-treatment group at 12 months or longer [58].Although many reports recommending surgery emphasize the side effects of DA treatment and the disadvantages of long-term use, it should be noted that the goal of treating prolactinomas, especially microprolactinomas, is to reverse gonadal dysfunction, not necessarily to wean the patient off of DA treatment. ",
"section_name": "Surgical Treatment",
"section_num": "6."
},
{
"section_content": "The aims of radiation therapy for prolactinomas are (1) to inhibit the growth of the tumor, (2) suppress hormone secretion, and (3) suppress the further progression of the tumor remnants that pathologically indicate aggressive behavior through adjuvant therapy.There are different varieties of radiotherapy.Fractionated external beam radiotherapy (EBRT), known as conventional radiotherapy, had been used mainly for pituitary tumors until recently.Now, stereotactic radiosurgery (SRS) or stereotactic radiotherapy (SRT) tends to be preferred because each allows the target zone to be irradiated while sparing the surrounding or peripheral healthy tissue.Hormone normalization with SRS takes less time [59].However, EBRT has also improved as an intensity-modulated radiotherapy (IMRT) [60]. SRS is an effective option for controlling the growth of residual or recurrent tumors after surgical resection, but the rate of endocrine improvement or remission is low in functioning PitNETs.In meta-analyses, SRS resulted in tumor control rates of 86-100%, and endocrine remission rates varied between 6 and 81%, with remission rates increasing over time [61]. The most common side effect associated with SRS is hypopituitarism, with the incidence of new hormone deficiencies observed at 4.5-42%.The reported incidence of hypopituitarism with EBRT is approximately 50% 10 to 20 years after treatment.Since one of the purposes of prolactinoma treatment is to restore gonadal function, radiation therapy may be contra-indicated if it causes central hypogonadism.New neurological or visual defect rates ranged from 0 to 5% after SRS [61,62]. ",
"section_name": "Radiation Therapy",
"section_num": "7."
},
{
"section_content": "Prolactinomas in young women often coincide with pregnancy and childbirth, and thus careful management is required.Although bromocriptine and cabergoline have been shown to have no adverse effects on the course of the pregnancy or the fetus, DAs should be discontinued in principle if pregnancy occurs during drug therapy [63,64].There were no abnormalities in maternal-fetal outcomes in 6272 pregnancies with bromocriptine or in 1061 with cabergoline [65].Regarding the risk of tumor growth during pregnancy, a systematic review indicated 2.4% symptomatic tumor growth in microprolactinomas, 21% in macroprolactinomas, and 4.7% in macroprolactinomas with previous surgery or radiotherapy [63].Since there have been a few cases of tumor growth during the interruption of DAs during pregnancy, the course of clinical symptoms (e.g., headache or visual field disturbances) should be carefully monitored [1,63,66].In healthy women, the high estrogen levels in pregnancy cause lactotroph hypertrophy and hyperprolactinemia, and serum PRL levels gradually elevate during the course of gestation; thus, serum PRL is not an indicator of prolactinoma progression.An MRI scan without gadolinium should be performed if a patient with macroprolactinoma has severe headaches or a visual field defect.DAs or transsphenoidal surgery should be considered if aggressive macroprolactinoma is evident [63,64]. ",
"section_name": "Pregnancy",
"section_num": "8."
},
{
"section_content": "A giant prolactinoma is defined as prolactinoma with a tumor >4 cm and very high serum PRL levels.It is a rare type of PitNET accounting for 0.5-4.4% of PitNETs and 2-3% of prolactinomas.The giant prolactinoma occurs predominantly in males, with a male to female ratio of 9:1 [67,68].Extremely high levels of PRL cause the \"hook effect\" [22]. As mentioned above, it has been reported that DA treatment for giant prolactinomas results in tumor shrinkage and an improved visual field: 90% showed tumor shrinkage alone, and PRL was also normalized in approximately 75-80% of cases with cabergoline treatment [67].Surgery alone rarely cures giant prolactinomas.Most patients do not achieve hormonal remission after surgery, and surgical mortality and morbidity are high [68].However, lifelong continuous DA therapy is required in almost all patients to maintain PRL suppression and prevent tumor regrowth [69]. Especially in giant prolactionomas, tumor shrinkage from DA treatment may rarely result in spontaneous CSF leakage.In such cases, bone defects of the cellular floor are seen on a CT scan due to tumor invasion [70]. ",
"section_name": "Giant Prolactinoma",
"section_num": "9."
},
{
"section_content": "PitNETs are defined as malignant only when distant metastases are present and are called \"pituitary carcinomas\".Malignant prolactinomas are reported to metastasize after a latency period of 2 months to 22 years, with an average of 4.7 years.The prognosis for pituitary carcinoma is poor, with an average survival of 10 months after diagnosis of metastasis.Moreover, the WHO classification defines an \"aggressive PitNET\" as having a radiologically invasive tumor and an unusually rapid tumor growth rate or clinically relevant tumor growth despite optimal standard therapies (surgery, radiotherapy, and conventional medical treatments).Although aggressive prolactinomas are essentially DAresistant, prolactinomas that are hormonally uncontrolled but without tumor progression should not be considered aggressive PitNETs [71,72].Prolactinomas are the second-most common tumor type treated with temozolomide after corticotroph pituitary tumors and represent about 30% of cases, and most DA-resistant prolactinomas are macrotumors.In a European Society of Endocrinology survey, 75% of aggressive PitNETs or pituitary carcinomas occurred in men [73,74]. Temozolomide is an alkylating agent that has shown efficacy in glioblastomas.Subsequently, temozolomide was reported to be effective against aggressive PitNETs and pituitary carcinomas (Figure 2).In 2017, the European Society of Endocrinology Clinical Practice Guidelines advised using temozolomide as a first-line chemotherapy treatment for aggressive PitNETs and carcinomas [74].A recent systematic review estimated the five-year overall survival at 57% for all patients treated with temozolomide [73].The response rate for prolactinomas and corticotroph tumors is about 50%, which compares favorably with nonfunctioning or GH-secreting tumors.After discontinuing temozolomide, the frequency of tumor regrowth ranged from 27 to 67%, with progression-free survival mainly in the range of 8 to 18 months.Even if pituitary tumors respond initially to temozolomide treatment, they often develop a resistance to it, so a second course of temozolomide treatment after regrowth is almost always ineffective [75]. pituitary carcinoma is poor, with an average survival of 10 months after diagnosis of metastasis.Moreover, the WHO classification defines an \"aggressive PitNET\" as having a radiologically invasive tumor and an unusually rapid tumor growth rate or clinically relevant tumor growth despite optimal standard therapies (surgery, radiotherapy, and conventional medical treatments).Although aggressive prolactinomas are essentially DA-resistant, prolactinomas that are hormonally uncontrolled but without tumor progression should not be considered aggressive PitNETs [71,72].Prolactinomas are the second-most common tumor type treated with temozolomide after corticotroph pituitary tumors and represent about 30% of cases, and most DA-resistant prolactinomas are macrotumors.In a European Society of Endocrinology survey, 75% of aggressive PitNETs or pituitary carcinomas occurred in men [73,74]. Temozolomide is an alkylating agent that has shown efficacy in glioblastomas.Subsequently, temozolomide was reported to be effective against aggressive PitNETs and pituitary carcinomas (Figure 2).In 2017, the European Society of Endocrinology Clinical Practice Guidelines advised using temozolomide as a first-line chemotherapy treatment for aggressive PitNETs and carcinomas [74].A recent systematic review estimated the five-year overall survival at 57% for all patients treated with temozolomide [73].The response rate for prolactinomas and corticotroph tumors is about 50%, which compares favorably with nonfunctioning or GH-secreting tumors.After discontinuing temozolomide, the frequency of tumor regrowth ranged from 27 to 67%, with progression-free survival mainly in the range of 8 to 18 months.Even if pituitary tumors respond initially to temozolomide treatment, they often develop a resistance to it, so a second course of temozolomide treatment after regrowth is almost always ineffective [75].O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes the alkylation adducts induced by temozolomide and counteracts the antitumor actions of temozolomide.In addition, mutations in mismatch repair proteins, particularly MSH6, independent of MGMT, increase temozolomide resistance.These can be evaluated by immunohistochemistry, and a good response to temozolomide was reported when MGMT immunostaining was negative.Low MGMT expression is more common in aggressive PitNETs and pituitary carcinomas than in non-aggressive PitNETs, and prolactinomas are more likely to have low MGMT expression than other subtypes [75,76].This patient was a 64-year-old man.Although he underwent transsphenoidal surgery three times and was treated with 7 mg/week cabergoline, the tumor progressed.After commencing temozolomide treatment, the tumor shrank markedly, and serum prolactin decreased to below the reference range.Temozolomide was withdrawn after 34 courses, and no tumor recurred over the next 3 years. O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes the alkylation adducts induced by temozolomide and counteracts the antitumor actions of temozolomide.In addition, mutations in mismatch repair proteins, particularly MSH6, independent of MGMT, increase temozolomide resistance.These can be evaluated by immunohistochemistry, and a good response to temozolomide was reported when MGMT immunostaining was negative.Low MGMT expression is more common in aggressive PitNETs and pituitary carcinomas than in non-aggressive PitNETs, and prolactinomas are more likely to have low MGMT expression than other subtypes [75,76]. ",
"section_name": "Pituitary Carcinoma and Aggressive Prolactinoma",
"section_num": "10."
},
{
"section_content": "Recently, the effectiveness of a new medical treatment using estrogen receptor (ER) antagonists such as tamoxifen or raloxifene was validated and showed promising results [77].Considering the fact that ER is expressed in most of the surgical prolactinoma specimens examined and that, as described above, estrogen facilitates lactotroph tumor cell growth, the addition of an ER antagonist to conventional dopamine receptor agonist is reasonable and awaits further study.More recently, the effectiveness of the mammalian target of rapamycin (mTOR) inhibitor or tyrosine kinase inhibitor has also been reported [78,79]. ",
"section_name": "New Therapeutic Approach",
"section_num": "11."
}
] |
[
{
"section_content": "Prolactinomas are a common disease among pituitary disorders, most of which can be treated by DAs; however, surgical treatment is required in some cases.Recently, the efficacy of temozolomide for malignant prolactinomas was reported. ",
"section_name": "Conclusions",
"section_num": "12."
},
{
"section_content": "Funding: This review received no external funding. The authors declare no conflict of interest. We searched the PubMed using combinations of the terms \"epidemiology\", \"pathogenesis\", \"diagnosis\", \"hyperprolactinemia\", \"medical treatment\", \"dopamine agonist\", \"surgical treatment\", \"radiation\", \"pregnancy\", \"giant\", \"pituitary carcinoma\", \"aggressive\", and \"prolactinoma\". ",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "Funding: This review received no external funding. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors declare no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "We searched the PubMed using combinations of the terms \"epidemiology\", \"pathogenesis\", \"diagnosis\", \"hyperprolactinemia\", \"medical treatment\", \"dopamine agonist\", \"surgical treatment\", \"radiation\", \"pregnancy\", \"giant\", \"pituitary carcinoma\", \"aggressive\", and \"prolactinoma\". ",
"section_name": "Search Strategies:",
"section_num": null
},
{
"section_content": "Author Contributions: The introduction and the sections on epidemiology, diagnosis, medical treatment and pregnancy were written by M.N.The sections on surgical treatment, radiation therapy, giant prolactinoma and pituitary carcinoma and aggressive prolactinoma were written by N.F.The section on pathogenesis and new therapeutic approaches was written by Y.I.All authors have read and agreed to the published version of the manuscript. ",
"section_name": "",
"section_num": ""
}
] |
10.1038/s41408-022-00684-8
|
Effectiveness, immunogenicity, and safety of COVID-19 vaccines for individuals with hematological malignancies: a systematic review
|
<jats:title>Abstract</jats:title><jats:p>The efficacy of SARS-CoV-2 vaccination in patients with hematological malignancies (HM) appears limited due to disease and treatment-associated immune impairment. We conducted a systematic review of prospective studies published from 10/12/2021 onwards in medical databases to assess clinical efficacy parameters, humoral and cellular immunogenicity and adverse events (AE) following two doses of COVID-19 approved vaccines. In 57 eligible studies reporting 7393 patients, clinical outcomes were rarely reported and rates of SARS-CoV-2 infection (range 0–11.9%), symptomatic disease (0–2.7%), hospital admission (0–2.8%), or death (0–0.5%) were low. Seroconversion rates ranged from 38.1–99.1% across studies with the highest response rate in myeloproliferative diseases and the lowest in patients with chronic lymphocytic leukemia. Patients with B-cell depleting treatment had lower seroconversion rates as compared to other targeted treatments or chemotherapy. The vaccine-induced T-cell response was rarely and heterogeneously reported (26.5–85.9%). Similarly, AEs were rarely reported (0–50.9% ≥1 AE, 0–7.5% ≥1 serious AE). In conclusion, HM patients present impaired humoral and cellular immune response to COVID-19 vaccination with disease and treatment specific response patterns. In light of the ongoing pandemic with the easing of mitigation strategies, new approaches to avert severe infection are urgently needed for this vulnerable patient population that responds poorly to current COVID-19 vaccine regimens.</jats:p>
|
[
{
"section_content": "The ongoing coronavirus disease 2019 (COVID-19) pandemic causes immense mortality and morbidity [1].Patients with hematological malignancies (HM) have a higher risk of infection after exposure, a worse prognosis after infection, and a higher risk of severe or critical disease and COVID-19-related complications due to disease and/or treatment associated with complex immune dysfunction [2][3][4][5]. Vaccines were shown to be a key element to prevent severe diseases resulting in hospitalization and death in most patients [6].Current evidence proves impaired vaccine-induced immune response in immunocompromised individuals with HM [7,8].At the same time, data regarding vaccination to mitigate sequelae by SARS-CoV-2 variants of concern (VOC) is indicative of reduced neutralizing activity and protective efficacy of vaccination, compared to the wild type or less pathogenic variants [9,10].Ongoing studies evaluating booster vaccinations and the aim to develop adjusted vaccines addressing VOCs are of utmost importance-especially when considering their impact on vulnerable patient groups, such as immunocompromised individuals [11]. COVID-19 vaccine immunity is mediated by the interplay of humoral and cellular components [12].In many HM patients a disease-induced dysfunction of the innate and adaptive immune system, as well as a treatment-related immune deficiency, severely impacts the immune response following COVID-19 vaccination.Mechanisms underlying protection against COVID- 19 are not yet fully understood.There is already strong evidence that the humoral response is required to prevent infection, while the cellular response seems to be critical for the prevention of severe disease.Yet, the humoral response may not always correlate with T-cell-mediated immunity in immunocompromised patients [13][14][15].Data evaluating the vaccine-induced immune response is generally scarce for HM patients since most clinical trials initially excluded such patients.In the past months, several studies have investigated vaccine-induced immunogenicity in patients with cancer and indicated a decreased immune response to COVID-19 vaccines.A better understanding of the immune response to SARS-CoV-2 vaccination in these individuals is critical for optimizing vaccination programs, identifying particularly vulnerable patient groups and areas of unmet need, and thereby reducing severe disease and mortality. As people with HM are at high risk of severe disease after infection with SARS-CoV-2 [16], and research on vaccine effectiveness in this population is generally scarce, there is an urgent ongoing need to evaluate the effectiveness, immunogenicity, and safety of COVID-19 vaccines for these individuals.There are several studies reporting on the effects of COVID-19 vaccines in hematological and oncological patients [17][18][19][20][21].However, a comprehensive synthesis of the available evidence and evaluation of patient-relevant outcomes is needed to support disease prevention and the identification of optimized vaccination schedules.Thus, the objective of this systematic review is to assess the effectiveness, immunogenicity, and safety of COVID-19 vaccines for individuals with HM. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "The protocol for this review was registered with PROSPERO (CRD42021281412) and is provided at Open Science Framework under https://osf.io/2nbev/. ",
"section_name": "METHODS",
"section_num": null
},
{
"section_content": "On December 10, 2021, the Web of Science Core collection, the WHO COVID-19 Global literature on Coronavirus disease, and Cochrane COVID-19 Study Register (CCSR)were searched.The search strategies can be found in the supplement.In addition, reference lists of included publications were hand searched. ",
"section_name": "Literature search",
"section_num": null
},
{
"section_content": "We included randomized controlled trials and prospective cohort studies, examining adult participants with a diagnosis of a HM who completed vaccination schedules with one or more of the COVID-19 vaccines that have been authorized for use in the European Union or approved, authorized, licensed or granted an emergency use authorization in at least ten countries worldwide; as of September 1, 2021; those were: mRNA-based vaccines: BNT162 (Comirnaty®) from Pfizer/Bion-techmRNA-1273 (Spikevax®) from Moderna; Vector-based vaccines: AZD1222 (Vaxzevria®) from Astrazeneca, JNJ-78436735 (COVID-19 Vaccine Janssen®) from Janssen Pharmaceuticals, rAd26 (Sputnik Light®) from Gamaleya, rAd5 (Sputnik V®, Gam-COVID-Vac®) from Gamaleya; Inactivated vaccines: BBIBP-CorV (Covilo®) from Sinopharm, CoronaVac (Sinovac®) from Sinovac Biotech [22]. Complete vaccination (=full primary immunization) schedules were defined as per marketing authorization at the time of study conduct and included two doses for full primary immunization (except COVID-19 Vaccine Janssen®).An additional dose at ≥3 months after completion of full primary immunization was considered as a booster dose.An additional dose <3 months after completion of full primary immunization was considered as optimization of the full primary immunization schedule. We did not exclude studies based on publication format, as long as sufficient information was available, and the abstract could be retrieved in English.Study selection was performed in duplicate using a web-based online platform (Rayyan; www.rayyan.ai)by VP, CH, AB, CI, and/or NK.Disagreements were resolved by discussion or by consulting a third author [23]. ",
"section_name": "Study selection criteria",
"section_num": null
},
{
"section_content": "Data were extracted by one reviewer and verified by another (VP, CH, CI, AB, NK, SM, and/or PJB).Predefined outcomes were clinical parameters (COVID-19 related mortality, COVID-19-related admission to intensive care unit (ICU), COVID-19 related hospitalization, symptomatic COVID-19, SARS-CoV-2 infection, time to infection, and transmissibility); immunity parameters describing the seroconversion and binding immunoglobulin G titers, seroconversion described by neutralization assays, and T-cell parameters; and adverse events, serious adverse events, and events of special interest (i.e., allergic reactions, thrombotic events, heart muscle inflammation, the progress of underlying malignancy). We collected outcome data (event frequencies and sample size) for the overall cohort and relevant subgroups and recalculated confidence intervals where necessary.Data on healthy controls of the same studies were extracted, but not directly compared to the HM cohorts, as most of these controls were not well-matched in age and clinical condition.Due to the large clinical, geographical, and temporal heterogeneity of studies, outcome rates were not pooled but depicted in form of forest plots without a combined effect estimate using R (version 4.1.2) as well as presented in a table format, both according to overall identified studies and subgroups (type of disease, type of therapy, age, the biological sex of participants). ",
"section_name": "Data extraction and synthesis of the evidence",
"section_num": null
},
{
"section_content": "For risk of bias assessments, we used a tool that is currently being developed for overall prognosis studies (RoB-OPS; https://osf.io/dfk2r)and which is based on previous items in prognosis and risk of bias research but tailored to studies without a control arm.Based on the four domains of participants, outcomes, analysis, and reporting bias, an overall judgement of each outcome set was made per study.The complete list of items and the rating options in their current form can be found in the supplement. For prioritized outcomes, we used the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach to assess the certainty in the evidence, starting at a high level of evidence according to guidelines for overall prognosis studies [24,25]. ",
"section_name": "Risk of bias assessment and GRADEing of the evidence",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "The search flow is summarized in Fig. 1.We initially identified 2575 potentially relevant records.After removing 326 duplicates and 1970 records through title and abstract screening, 279 full-text manuscripts were evaluated for eligibility.Further, 128 records were excluded for specific reasons (see Supplementary Table 1).Finally, 156 studies (151 records) that met the inclusion criteria were included.Of those, 58 studies were still ongoing or completed, but without published data (see Supplementary Table 2).After all, 57 studies were included in our outcome synthesis. ",
"section_name": "Results of the search",
"section_num": null
},
{
"section_content": "Of the 57 identified studies, all were conducted between December 2020 and October 2021.Most studies were conducted in Europe (29 of 57 studies), followed by 14 studies from the US, and 12 from Israel.We did not identify any information on study location for the two studies.The majority of studies received funding from academic organizations, charities or foundations, or governmental institutions (29 of 57 studies).Eight studies received no dedicated funding, one study was funded by the pharmaceutical industry and the remaining did not disclose funding sources. Ten studies included only individuals with plasma cell neoplasms (primarily multiple myeloma, MM), four only individuals with chronic lymphocytic leukemia (CLL), and one only individual with Waldenström macroglobulinemia.The remaining studies included participants with different malignancies or did not specify the underlying type of HM.In total, 39 studies had a control group, with 15 of those consisting of healthy volunteers, 12 healthy health care workers (HCW), 3 age-matched or -compatible cohorts, and 2 age-and sex-matched HCW.In two studies the control group consisted of a solid tumour cohort and a non-vaccinated COVID-19-positive MM patient cohort, respectively. Overall, we identified studies on five different vaccines, which were examined in various combinations: Comirnaty®, Spikevax®, Vaxzevria®, COVID-19 Vaccine Janssen®, and BBIBP-CorV, with Comirnaty® used as a single vaccine in 31/57 studies.Most studies evaluated the effectiveness of a defined full primary immunization schedule (i.e., one dose for COVID-19 Vaccine Janssen®, and two doses for all other vaccines).Two studies evaluating the effect of a third vaccine dose were identified.None of the studies had a prospectively planned interventional comparison.However, five studies enrolled participants receiving different vaccines and provided subgroup outcome data. All of the included studies reported on at least one immunity parameter of interest.Clinical outcomes evaluating the effectiveness of the vaccines were reported in 22 of 57 studies, and safety outcomes, including data on reactogenicity, were reported in 20 of the studies.Supplementary Table 3 summarizes details regarding the study and participant characteristics. ",
"section_name": "Description of studies",
"section_num": null
},
{
"section_content": "Effectiveness outcomes.From the 22 studies reporting clinical outcomes, one study was separately rated for three different clinical outcomes.Among these 24 judgments, the overall risk of bias was rated to be low in three studies, to be moderate in ten studies and to be high in 11 studies.For nine studies no information was available to assess selective reporting bias.The separate judgements for each domain are illustrated in Supplementary Fig. 1, and supporting judgements are available from the authors upon request. Immunogenicity outcomes.From the 57 studies reporting immunogenicity outcomes, three studies were separately rated for two different immunogenicity outcomes.Among these 60 judgements, the overall risk of bias was rated to be low in ten studies, to be moderate in 36 studies and to be high in 14 studies.For nine studies no information was available to assess selective reporting bias.The separate judgements for each domain are illustrated in Supplementary Fig. 2. Safety outcomes.From the 20 studies reporting safety outcomes, one study was separately rated for two different safety outcomes.Among these 21 judgements, the overall risk of bias was rated to be low in none of the studies, to be moderate in nine studies and to be high in 12 studies.For seven studies no information was available to assess selective reporting bias.The separate judgements for each domain are illustrated in supplementary fig. 3. Effectiveness, immunogenicity, and safety of vaccination.A summary of findings with the GRADE-certainty assessments, for outcomes prioritized as most patient-relevant at the protocol stage, is available in Table 1.Further, an overview of all reported outcomes, including the ranges of reported effect rates, and the number of participants and studies is available in Supplementary Table 6.SARS-CoV-2 infection and severity: We identified 19 studies (3277 participants with HM) reporting on SARS-CoV-2 infections following full primary immunization.The event rate ranged across studies from 0 to 11.9% (⊕⊖⊖⊖ very-low certainty of the evidence) (see Fig. 2A).The time of follow-up (FU) was not reported in six studies [26][27][28][29][30][31], and ranged from 11 days to 6 months after the second vaccination in the remaining studies.One study (72 participants with HM) reported the time to occurrence of SARS-CoV-2 infections after full primary immunization.The study reported two cases that occurred after a median 19.5 days (range 16-23) following the second vaccination.It was not reported whether both individuals achieved an immune response after vaccination. We identified 13 studies (1484 participants with HM) reporting on symptomatic COVID-19.The event rate ranged across studies from 0 to 2.7% (⊕⊖⊖⊖ very-low certainty of the evidence) (see Fig. 2B).FU time was not reported in six studies [26][27][28][29][30][31] and ranged from 19 days to 6 months after the second vaccination in the remaining studies. Six studies (382 participants with HM) reporting on COVID-19 related hospitalization (Mona 2021) were identified.The event rate ranged studies from 0 to 2.8% across studies (⊕⊖⊖⊖ very-low certainty of the evidence) (see Fig. 2C).FU time was not reported in three studies [28,32,33] one had a follow-up of 2 months, and two studies of 3 months [34,35] after the second vaccination.One study (72 participants with HM) reported on COVID-19 related admission to ICU.After a follow-up of 3 months after the second vaccination, no cases were observed [35]. In total, we identified six studies (1228 participants with HM) reporting on COVID-19-related mortality in vaccinated individuals.The event rate ranged across studies from 0 to 0.5% (⊕⊖⊖⊖ verylow certainty of the evidence) (see Fig. 2D).We did not identify any studies reporting on transmissibility (e.g., secondary attack rates) or quality of life after SARS-CoV-2 vaccination. ",
"section_name": "Risk of bias",
"section_num": null
},
{
"section_content": "Humoral response: Forty-eight studies reported on antibody response after full primary vaccination.Of those, 18 compared their results to a healthy control group and described significant between-group differences in antibody response.Positive humoral immune response was defined in the primary studies according to cutoffs defined for the respective assays used (see Supplementary Table 6).Across studies (including 7393 individuals), response rates ranged from 38.1 to 99.1% after the first measurement at least 14 days after the second vaccine (⊕⊕⊖⊖ low certainty of the evidence) (see Fig. 2E).We stratified the results along underlying disease and treatment received (see Table 2).Detailed results with the number of patients and events are depicted in the corresponding forest plots Supplementary Figs. 4 and5, respectively.The highest response rate was seen for patients with myeloproliferative diseases and the lowest in patients with CLL.Patients with lymphoma showed very diverse responses irrespective of whether indolent or aggressive lymphoma was reported.Regarding treatment, patients with B-cell depleting or -directed treatment had lower seroconversion rates as compared to those receiving chemotherapy or other targeted treatments.Patients who underwent either allogeneic or autologous stem cell transplantation (SCT) showed higher seroconversion rates than those after CAR T-cell therapy.Available data differed in quantity and detail with regard to different underlying diseases (Fig. 3A).Most data were available for patients with indolent lymphoma, especially MM, and CLL, while data for patients with acute leukaemia and myeloproliferative diseases are sparse.Due to different assays applied in the various studies including heterogeneity in target measurement throughout the studies, the direct comparison of IgG titers was unfeasible and is not reported. We identified five studies reporting on FU after the second vaccine dose.During the initial measurement, 573 participants with HM were evaluated while at the extended FU measurement 373 participants were evaluated.Response rate at the initial measurement ranged from 58 to 82% and after the extended FU from 45 to 76%, with four studies suggesting a waning IgG antibody response over time (Supplementary Fig. 6).Measurements were performed ~2-3.5 months apart. Two studies reported IgG response after a third (i.e., \"booster\") vaccine dose for a total of 78 participants with HM.The response rate ranged from 31 to 65%.For one study, IgG measurements after the second dose were also available, however for a larger cohort.In comparison, the response rate for the full cohort was 75% after completing full primary immunization and 65% for the patients receiving a booster dose (Supplementary Fig. 7) [36]. Five studies (429 participants with HM) compared IgG antibody responses for different vaccine types.Four studies compared BNT162b2 with mRNA-123 [30,[37][38][39] and one study BNT162b2 with ChAdox-nCoV-19 [40].Confidence intervals were widely overlapping for all but one study that compared BNT162b2 with mRNA-123 (27% (95% CI 15-40) vs. 62% (95% CI 49-76)).Further, the one study comparing BNT162b2 with ChAdox-nCoV-19 suggests no difference in response (supplementary fig.8). Eighteen studies provided data on healthy controls.As described above, no direct comparisons to the HM cohorts were made, as most of these controls were not well-matched in age and clinical condition.Seroconversion in healthy controls ranged from 96 to 100% (supplementary fig.9). Of all studies searched, 14 reported the development of neutralizing antibodies against SARS-CoV-2 (1174 participants with HM).Response rate ranged across studies from 21.8 to 96.2% after the first measurement at least 14 days after the second vaccine (see supplementary table 7). Cellular response: There were 11 studies reporting vaccineinduced T-cell responses in patients with HM.To evaluate such response, different assays were used across the studies and T-cell immunity was analyzed by intracellular cytokine staining and flow cytometry, measurement of secreted interferon-gamma (IFN-γ) by the ELISpot technique, or a whole-blood platform, and by measurement of secreted IFN-γ/Interleukin (IL)-2 by the Fluoro-Spot technique.T-cell response rates ranged from 26.5 to 85.9% in all studies (⊕⊕⊖⊖ low certainty of the evidence) (see Fig. 2F).Two studies providing data for HSCT patients only, reported the lowest response rates of 27% and 29%, respectively [41,42].Five studies did not further specify subgroups of hematological patients, three studies reported on multiple myeloma patients only [43][44][45], and the remaining study included various types of lymphoma [46].The timepoint of T-cell measurement ranged between 14 days and 6 weeks after full primary immunization (two vaccines).Four studies further specified their results on the type of treatment with very small sample sizes in consequence.Yet, patients with anti-CD20 therapy within 6 months from vaccination were shown to have robust T-cell responses in Safety.We identified six studies (853 participants with HM) reporting on adverse events (AEs).Across studies 0-50.9% of participants experienced at least one event (⊕⊕⊖⊖ low certainty of the evidence) (see Fig. 2G).Three studies did not report the observation period [28,49,50].In addition, 17 studies reported local and/or systemic reactions or AEs of special interest for their study; an overview of reported events can be found in Supplementary Table 8.We identified three studies (208 participants with HM) reporting on vaccine-induced immune thrombotic thrombocytopenia (VITT).Across studies, 0-2.5% of participants experienced at least one event.One study did not report the observation period [37], one study observed participants for 49 days from the first dose [27], and one study for 2 weeks after the second dose [31].Three studies (761 participants with HM) reported anaphylactic or systemic allergic reactions, 0-1.3% of participants experienced at least one event.Two studies (226 participants with HM) reporting the progress of the underlying HM were identified and 2.5-5.5% of participants reported experiencing progression of their underlying disease.Participants were followed for up to 1 month after the second vaccine dose; however, we did not identify any information on whether the disease progression was likely related or unrelated to the vaccination.We did not identify any studies reporting any events of myocarditis or termination of anti-cancer treatment due to vaccination.Four studies (901 participants with HM) reported serious adverse events (SAEs).Across studies, 0-7.5% of participants experienced at least one event (⊕⊕⊖⊖ low certainty of the evidence) (see Fig. 2G).SAEs were assessed for at least 7 and up to 49 days following vaccination. ",
"section_name": "Immunogenicity",
"section_num": null
},
{
"section_content": "We here report the results of a systematic review assessing the effectiveness, immunogenicity, and safety of COVID-19 vaccines in patients with HM.Clinical outcomes assessing vaccine effectiveness were generally only occasionally and not systematically reported and further limited by short observational periods.Thus, a firm appraisal of the protective effect of COVID-19 vaccines on these highly patient-relevant outcomes is to date not possible.With regard to immunogenicity, we found heterogeneous vaccine-induced seroconversion rates and cellular immunity in HM patients, which was lower than reported for healthy participants in all studies.The highest humoral response rates were reported for myeloproliferative diseases and the lowest in CLL.Poor immunogenicity by COVID-19 vaccines in patients with an impaired B-cell axis is in line with other vaccines showing reduced efficacy in this population [51][52][53][54][55]. The detrimental effect of B-cell depleting treatment, especially when administered within 12 months from vaccine application, is concerning beyond the population of HM patients studied herein since these drugs are widely used beyond HM [56,57].Interestingly, initiating B-cell depleting treatment shortly after vaccination does not seem to relevantly impair humoral vaccine response [58].The relatively low humoral response to vaccination in patients undergoing CAR-T-cell treatment compared to HSCT recipients could be attributable to the lymphodepleting chemotherapy and B-cell targeting constructs.Additionally, a historic effect, with HSCT recipients potentially recovered from treatment effects, appears plausible [59].Due to different assays used and lacking international standards, a pooled outcome analysis of antibody titres was infeasible.However, it is generally questionable whether the serologic response can serve as reliable CoP in a population with an impaired B-cell axis. In contrast to the serologic response to COVID-19 vaccines, insights into cellular vaccine response are still scarce.In the light of impaired serologic response in HM, SARS-CoV-2 specific T cells may be crucial in this population.Of 57 studies included, only 11 reported vaccine-induced T-cell response.Except for patients after allogeneic HSCT, robust T-cell responses were reported in HM patients across the studies included.Especially those receiving anti-CD20 treatment responded well on the T-cell level highlighting the protective capacities of vaccination also in patients with treatment-associated B cells [60].Studies reporting on discordant immune responses show that at least 20% of patients show a T-cell response in the absence of seroconversion.These results are reassuring for those patients failing serologic response but question the role of antibodies as the sole correlate of protection.Caution is warranted; however, since most studies used different assays, different thresholds and usually lacked control groups.Further, the kinetics of T cells after COVID-19 vaccination in the immunocompromised remain sparsely investigated, and therefore, the ideal timepoint for T-cell measurement is yet to be determined. With regard to safety, AE assessment and reporting were not performed systematically in most studies.However, reported events herein are largely consistent with the safety profile observed in clinical trials and real-world evidence of the general population [61][62][63].AEs of special interest as identified and prioritized in the conducted patient workshop were rarely or never reported. Given the rapid changes in the epidemiological situation and turnover of research output in the SARS-CoV-2 / COVID-19 field, it is nearly impossible to stay up to date with a methodologically sound and systematic approach.The problem of waning immunity was identified only in the last months and optimization schedules are widely investigated and partially already applied through an additional booster dose.A recent systematic review with metaregression provides evidence of a decrease in vaccine effectiveness after full primary immunization against pre-omicron SARS-CoV-2 variant infection by 21% (95% CI 13.9-29.8) in the general population [64].By the time of our search, we only identified a small number of studies investigating a third vaccine dose in HM patients.Interestingly, a more recent study reported successfully seroconversion after a third mRNA vaccine dose in 59% of 75 patients that had received cellular therapies or bispecific antibodies and failed the primary two-dose vaccine scheme [65].This and other emerging studies highlight the potential of booster vaccinations and several international committees on immunization already recommend a fourth vaccine dose for immunologically vulnerable patient groups [66,67]. None of the identified studies investigated vaccine effectiveness against the omicron VOC (BA.1, BA.2).First studies investigating neutralizing effectiveness against omicron in cancer patients suggest a higher immune evasive capacity; in individuals with HM also after receiving the third dose [68].Even though the evidence synthesized here does not assess evidence on variantspecific vaccine effectiveness or effectiveness of (repeated) boosters, it provides a thorough summary of the capacity to develop a humoral and/or cellular response following vaccination for individuals with different HM stratified by disease entity and treatments received.Taken together, we identified growing evidence for humoral COVID-19 vaccine-induced humoral response, but scarce data for a cellular response, and even less evidence for clinical vaccine effectiveness and safety in HM patients.To date, patients with MM or CLL and those receiving B-cell depleting therapies are the best-described patient groups with regard to humoral response.In contrast, data for patients with other diseases such as acute leukemias or aggressive lymphoma are still largely lacking.As highlighted by the evidence-gap maps (Fig. 3) a large evidence gap remains for most HM patients and underlying treatment and/or disease status, while data on the cellular immune response is particularly scarce. Overall, our systematic review highlights the generally poor immune response of people with HM to COVID-19.In light of easing or ending strategies aimed to reduce virus transmission for the general population, the flux in prevalent SARS-CoV-2 variants and entailing high incidence rates pose major risks to this vulnerable group.For high-risk individuals who respond poorly to current COVID-19 vaccination regimens, new approaches to prevent infections and severe or prolonged disease courses are urgently needed. Further research should therefore evaluate improved booster immunization strategies including heterologous vaccinations for immunocompromised individuals, such as the herein reported group of HM patients.By defining standards for reporting vaccine efficacy on the humoral and cellular level and enforcing the adequate reporting of clinical outcomes, international boards of experts could guide future research to enhance cross-study comparability and maximize the evidence and benefit generated. ",
"section_name": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "This systematic review was funded by the Federal Ministry of Education and Research in Germany (Grant no: 01KG2104).PJB is supported by Deutsche Krebshilfe through a Mildred Scheel Nachwuchszentrum Grant (no.70113307).Open Access funding enabled and organized by Projekt DEAL. ",
"section_name": "FUNDING",
"section_num": null
},
{
"section_content": "The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. ",
"section_name": "DATA AVAILABILITY",
"section_num": null
},
{
"section_content": "Further information on research design is available in the Nature Research Reporting Summary linked to this article. VP, AB, CH, CI, AA, IM, NK, JS, PJB, and NS declare no financial conflicts of interest with regard to the present study.SCM reports grants from DZIF.OAC reports grants or contracts from Amplyx, Basilea, BMBF, Cidara, DZIF, EU-DG RTD (101037867), F2G, Gilead, Matinas, MedPace, MSD, Mundipharma, Octapharma, Pfizer, Scynexis; Consulting fees from Amplyx, Biocon, Biosys, Cidara, Da Volterra, Gilead, Matinas, MedPace, Menarini, Molecular Partners, MSG-ERC, Noxxon, Octapharma, PSI, Scynexis, Seres; Honoraria for lectures from Abbott, Al-Jazeera Pharmaceuticals, Astellas, Grupo Biotoscana/United Medical/Knight, Hikma, MedScape, MedUpdate, Merck/MSD, Mylan, Pfizer; Payment for expert testimony from Cidara; Participation on a Data Safety Monitoring Board or Advisory Board from Actelion, Allecra, Cidara, Entasis, IQVIA, Jannsen, MedPace, Paratek, PSI, Shionogi; A patent at the German Patent and Trade Mark Office (DE 10 2021 113 007.7);Other interests from DGHO, DGI, ECMM, ISHAM, MSG-ERC, Wiley. The online version contains supplementary material available at https://doi.org/10.1038/s41408-022-00684-8. Correspondence and requests for materials should be addressed to Nicole Skoetz. 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": "Reporting summary",
"section_num": null
},
{
"section_content": "Further information on research design is available in the Nature Research Reporting Summary linked to this article. ",
"section_name": "Reporting summary",
"section_num": null
},
{
"section_content": "",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "VP, AB, CH, CI, AA, IM, NK, JS, PJB, and NS declare no financial conflicts of interest with regard to the present study.SCM reports grants from DZIF.OAC reports grants or contracts from Amplyx, Basilea, BMBF, Cidara, DZIF, EU-DG RTD (101037867), F2G, Gilead, Matinas, MedPace, MSD, Mundipharma, Octapharma, Pfizer, Scynexis; Consulting fees from Amplyx, Biocon, Biosys, Cidara, Da Volterra, Gilead, Matinas, MedPace, Menarini, Molecular Partners, MSG-ERC, Noxxon, Octapharma, PSI, Scynexis, Seres; Honoraria for lectures from Abbott, Al-Jazeera Pharmaceuticals, Astellas, Grupo Biotoscana/United Medical/Knight, Hikma, MedScape, MedUpdate, Merck/MSD, Mylan, Pfizer; Payment for expert testimony from Cidara; Participation on a Data Safety Monitoring Board or Advisory Board from Actelion, Allecra, Cidara, Entasis, IQVIA, Jannsen, MedPace, Paratek, PSI, Shionogi; A patent at the German Patent and Trade Mark Office (DE 10 2021 113 007.7);Other interests from DGHO, DGI, ECMM, ISHAM, MSG-ERC, Wiley. ",
"section_name": "COMPETING INTERESTS",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https://doi.org/10.1038/s41408-022-00684-8. Correspondence and requests for materials should be addressed to Nicole Skoetz. 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
}
] |
10.1038/s41467-020-17917-8
|
Genomic characterization of malignant progression in neoplastic pancreatic cysts
|
<jats:title>Abstract</jats:title><jats:p>Intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs) are non-invasive neoplasms that are often observed in association with invasive pancreatic cancers, but their origins and evolutionary relationships are poorly understood. In this study, we analyze 148 samples from IPMNs, MCNs, and small associated invasive carcinomas from 18 patients using whole exome or targeted sequencing. Using evolutionary analyses, we establish that both IPMNs and MCNs are direct precursors to pancreatic cancer. Mutations in <jats:italic>SMAD4</jats:italic> and <jats:italic>TGFBR2</jats:italic> are frequently restricted to invasive carcinoma, while <jats:italic>RNF43</jats:italic> alterations are largely in non-invasive lesions. Genomic analyses suggest an average window of over three years between the development of high-grade dysplasia and pancreatic cancer. Taken together, these data establish non-invasive IPMNs and MCNs as origins of invasive pancreatic cancer, identifying potential drivers of invasion, highlighting the complex clonal dynamics prior to malignant transformation, and providing opportunities for early detection and intervention.</jats:p>
|
[
{
"section_content": "ancreatic cancer is a deadly disease with a dismal prognosis that is predicted to soon be the second leading cause of cancer death in the United States 1 .However, like other epithelial malignancies, pancreatic cancer arises from noninvasive precancerous lesions that are curable if detected and treated early enough.Although the majority of pancreatic cancers are believed to originate in microscopic precancerous lesions (pancreatic intraepithelial neoplasia or PanIN), a significant minority arise in association with larger cystic neoplasms that can be detected using currently available imaging technologies 2 .These neoplasms, which include intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs), are frequently diagnosed incidentally on abdominal imaging, identifying a cohort of at-risk patients with an important opportunity for prevention of invasive pancreatic cancer 2 .However, prevention must be balanced with potential overtreatment of low-risk lesions, as pancreatic resection carries significant morbidity and even occasional mortality 3 .There is a critical need to understand the molecular alterations that are associated with the development of invasive cancer, as these represent potential biomarkers to identify cysts at high risk for progression to carcinoma and thus requiring clinical intervention. Although genomic analyses have been performed on hundreds of invasive pancreatic cancers, relatively few noninvasive neoplasms have been analyzed comprehensively.Whole exome and targeted sequencing of small cohorts of IPMNs and MCNs have revealed driver genes characteristic of each type of cystic neoplasm [4][5][6] , while targeted analyses in larger cohorts have confirmed the prevalence of specific driver gene mutations that correlate with grade of dysplasia or histological subtype 7 .These studies have confirmed that hotspot mutations in the oncogenes KRAS and GNAS occur in low-grade lesions while mutations in other driver genes, including CDKN2A, TP53, RNF43, and SMAD4, occur with increasing prevalence in lesions with highgrade dysplasia or associated invasive carcinoma 8 .Targeted next generation sequencing has been used to analyze pancreatic driver genes in different regions of IPMNs, revealing a surprising degree of intratumoral genetic heterogeneity, even with respect to wellcharacterized driver gene mutations [9][10][11][12] .However, the above analyses were based on studies of either single regions from each neoplasm or a limited number of genes from multiple regions, and did not provide an analysis of the evolutionary relationship between different regions of pancreatic cysts and associated cancers.These limitations highlight the need for comprehensive genomic analysis of these cysts and associated invasive cancers to understand the molecular alterations that underlie the transition to invasive carcinoma. In this study we perform whole exome sequencing of IPMNs and MCNs and their associated invasive carcinomas.Importantly, we focus our study on small invasive carcinomas (<2.5 cm) in order to more precisely analyze the genetic alterations that occur at malignant transformation in pancreatic tumorigenesis.In addition, in a subset of our samples, we perform deep targeted next generation sequencing on a larger set of additional tissue samples in order to assess mutated loci through entire neoplasms, including areas of low-grade dysplasia, high-grade dysplasia, and invasive carcinoma.These analyses reveal important features of pancreatic tumorigenesis, including evolutionary relationships between different regions within cystic neoplasms as well as molecular alterations that may drive the transition from a noninvasive precursor lesion to invasive cancer. ",
"section_name": "P",
"section_num": null
},
{
"section_content": "Overall approach.In order to dissect the molecular relationships between non-invasive dysplastic lesions and invasive pancreatic cancers, we performed whole exome sequencing of 39 neoplastic tissue samples from 18 patients with small invasive carcinomas (<2.5 cm) associated with neoplastic pancreatic cysts, including 16 patients with IPMNs and 2 patients with MCNs (Supplementary Data 1).Whole exome sequencing was performed on one sample from the noninvasive component with high-grade dysplasia and one sample from the invasive cancer in each case, and for three cases an additional noninvasive sample with lowgrade dysplasia was also analyzed by whole exome sequencing.Matched normal samples were analyzed by whole exome sequencing in each case to exclude germline variants and to identify somatic mutations.Whole exome sequencing was performed with an average total coverage of 177× (distinct coverage of 145×), generating 1.3 TB of sequencing data (Supplementary Data 2). In addition to whole exome analyses, we performed targeted next generation sequencing of 109 microdissected tissue samples from seven of the above cases (six IPMNs and one MCN).For these targeted analyses, we performed laser capture microdissection to isolate neoplastic cells from every available tissue block of the noninvasive cyst and cancer specimens.Separate samples were microdissected based on grade of dysplasia, cell morphology, architecture, and spatial location.This resulted in 8-22 additional samples per case.The targeted panel analyses included all mutated loci identified in the whole exome sequencing of these seven cases, as well as the entire coding regions of 15 wellcharacterized pancreatic driver genes (Supplementary Data 3).The targeted sequencing had an average coverage of 508× (distinct coverage of 460×) (Supplementary Data 2). We developed an integrated mutation calling pipeline to rigorously assess mutations in all sample types in our analyses in order to confidently identify even subclonal alterations in samples with low neoplastic purity (see \"Methods\") (Fig. 1, Supplementary Data 4).In addition, we utilized both on target and off target reads to examine focal copy number changes as well as loss of heterozygosity throughout the genome (Fig. 2, Supplementary Data 5 and 6).From our whole exome sequencing analyses, we identified an average of 66 somatic mutations in samples from noninvasive components (range 26-111) and an average of 65 somatic mutations in invasive carcinoma samples (range 31-105) (Fig. 1a, Supplementary Data 4).An average of 47 somatic mutations were shared between the noninvasive and invasive components, while 19 somatic mutations were unique to samples from noninvasive components and 20 somatic mutations were unique to samples from invasive cancer (Fig. 1a).We also identified an average of five shared copy number alterations between noninvasive and invasive components, as well as an average of one copy number alteration unique to samples from invasive cancer.A similar mean proportion of somatic mutations and copy number alterations were unique to invasive samples (0.28 for somatic mutations, 0.34 for copy number alterations). Analysis of our combined whole exome and targeted sequencing data provided multiple insights into IPMN and MCN tumorigenesis.In every analyzed case, there were multiple shared mutations between the noninvasive and invasive components.These included both driver and passenger mutations, indicating that they shared a common phylogenetic ancestor (Fig. 1a,b).In addition, accumulation of unique mutations in both noninvasive and invasive components demonstrated independent evolution after the divergence of the subclone that gave rise to the invasive cancer (Fig. 3, Supplementary Figs.S1-S18).Analysis of additional adjacent low-grade or high-grade samples from the same lesions revealed a subset of shared mutations, suggesting that these dysplastic lesions preceded the development of the invasive carcinoma (Fig. 3, Supplementary Figs.S1, S2, S3, S5, and S16).Evolutionary analyses showed a branched phylogeny in Mutations in some genes (such as KRAS) were always shared, while others were enriched in samples from noninvasive (RNF43) or cancer (SMAD4).Fig. 3 Somatic mutations identified in MTP19 and MTP8 in targeted and whole exome sequencing.We show mutations identified in each sample, including low-grade IPMN (light blue), high-grade IPMN (dark blue), ductal cancer (red), mucinous cancer (pink), and cancerization (purple).The type of sequencing analysis (targeted or whole exome) performed for each sample is indicated in a track on the bottom.Representative images of neoplastic tissue stained by hematoxylin and eosin, as well as isolated regions before and after laser capture microdissection are shown for MTP19 and MTP8.each case, with multiple clonally related but distinct dysplastic samples from each neoplasm (Fig. 4).Importantly, removal of driver gene mutations from these analyses did not significantly alter the resulting phylogenies, indicating that the evolutionary relationships are supported by mutations in addition to driver alterations (which might be shared by chance).Thus, the inferred evolutionary relationships between IPMN/MCNs and cancer samples are robust, as the probability of sharing a non-hotspot mutation due to chance alone is vanishingly small (1 in ~50 million), while the mean number of shared point mutations was 47 (the vast majority of which did not occur in hotspots). Another potential explanation for shared mutations in noninvasive and invasive samples is the presence of a small number of cancer cells (contamination) in IPMN/MCN samples.Our detailed pathological characterization and macro-or microdissection minimized the risk of this sample impurity.In addition, variant allele frequencies (VAFs) of shared mutations in IPMN/MCN and cancer samples can also help to evaluate the likelihood of such contamination.In mutations shared between the IPMN/MCN and cancer, the mean VAF in the noninvasive samples was 0.38, with only 4% of shared mutations having a VAF below 0.1 in the noninvasive samples.These high VAFs indicate that the shared mutations were not the result of a small number of cancer cells contaminating the noninvasive samples.Overall, these data provide evolutionary evidence that IPMNs and MCNs were precursors to invasive pancreatic cancer, with lowgrade regions usually preceding high-grade regions and ultimately resulting in invasive carcinoma. Driver genes of IPMN/MCN tumorigenesis.Through whole exome and targeted sequencing analyses of 18 IPMNs/MCNs and associated invasive carcinomas, we confirmed the high prevalence of mutations in previously identified pancreatic driver genes, including mutations of KRAS (89% of cases), GNAS (28%), CDKN2A (44%), TP53 (67%), SMAD4 (50%), and TGFBR2 (17%) (Fig. 1b).Somatic mutations were also identified in RNF43 (56%), which has been previously highlighted for its role as a driver in mucin-producing pancreatic cysts 4 .Somatic mutations were observed at low prevalence in key positions in the PI3K (PIK3CA, TSC2) and WNT (APC, CTNNA2, CTNNB1) signaling pathways as well as in STK11 (Fig. 1b, Supplementary Data 4).Alteration of these genes and pathways has been previously reported in a fraction of IPMNs 4,7,13,14 .The two MCNs analyzed were similar to the IPMNs in the cohort, with hotspot mutations in KRAS, homozygous deletion of CDKN2A, and inactivating mutations in RNF43, among others, but as expected these MCNs did not have GNAS alterations (Supplementary Figs.S3, S7) 6 . In addition to driver genes previously reported in IPMNs, our data provide an opportunity to discover novel drivers of IPMN tumorigenesis.We identified somatic mutations in the DNA damage response gene ATM in 17% of lesions, including one nonsense mutation (Fig. 1b, Supplementary Data 4).In addition we identified alterations in Hedgehog pathway member GLI3 in 5 of 18 cases (28%) (Fig. 1b, Supplementary Data 4).We also identified somatic mutations in a previously described hotspot in SF3B1, which encodes a protein critical for RNA splicing (Fig. 1b, Supplementary Data 4).Amplifications of the well-characterized driver genes ERBB2 and MYC were each observed in a single case and have not been previously reported in IPMNs (Supplementary Data 5).Other altered genes with a previously unknown role in IPMN tumorigenesis include MUC16 (four cases), PTPRT (four cases), and CNTN5 (three cases) (Fig. 1b).Intriguingly, in one case, an STK11 mutation was found in combination with biallelic ATM loss and cancer-specific biallelic KEAP1 loss-the In all cases, noninvasive samples (blue/green) precede invasive samples (red/pink) in the evolutionary history.In MTP5, different invasive cancer samples are placed in different regions of the phylogeny, highlighting multiple independent invasion events in this lesion.In MTP19, a sample of cancerization (purple) has descended from invasive cancer samples. combination of these three mutations has previously been reported in lung cancers (Supplementary Fig. S3) 15 . Although KRAS mutations occur in the majority of IPMNs and are thought to initiate tumorigenesis in these lesions, two IPMNs lacked mutations in this gene.One case contained a hotspot mutation in codon 227 of GNAS, another potential initiator of IPMN tumorigenesis, as well as alterations in TP53 and RNF43 (Supplementary Fig. S15).The other case lacked mutations in any of the frequently altered pancreatic driver genes but contained hotspot mutations in both CTNNB1 (S45P) and SF3B1 (H662Q) (Supplementary Fig. S10).These cases highlight alternative pathways of initiation and progression in IPMNs lacking KRAS mutations. Order of genetic alterations in IPMN/MCN tumorigenesis.Our multiregion sequencing approach of IPMNs/MCNs and associated invasive carcinomas provided insights into the order of specific genetic alterations in pancreatic tumorigenesis.In 17 of the 18 cases, at least one somatic mutation in the initiating driver genes KRAS and GNAS was shared between the noninvasive component and associated invasive cancer, with the remaining case lacking mutations in these genes.Somatic mutations in TP53 and CDKN2A were also shared in the noninvasive component and associated invasive cancer in the majority of cases.In contrast, SMAD4 had alterations confined to the invasive carcinoma in three cases and was shared between noninvasive and invasive samples in four cases (Fig. 1b).The majority of SMAD4 alterations in all sample types were bi-allelic, including 5/7 in noninvasive samples and 6/7 in invasive samples (Supplementary Data 7).Alteration of TGFBR2, which functions in the same signaling pathway as SMAD4, was also restricted to the cancer in one case (Fig. 1b).The other genes with mutations restricted to the invasive cancer (CDKN2A, CNTN5, PIK3CA, KEAP1, and RET) only had this pattern in a single sample (Fig. 1b). Our study also identified driver mutations in subclones of noninvasive neoplasms that diverged from and were not present in invasive cancer.These included hotspots mutations in wellcharacterized oncogenes and inactivating mutations in tumor suppressor genes (e.g., PIK3CA p.E545K, CTNNB1 p.S45F, SMAD4 p.E33fs, and multiple inactivating RNF43 mutations in patient MTP3) (Supplementary Fig. S3).Mutations in RNF43 were a particularly striking finding in these cases, as some noninvasive components contained several different RNF43 mutations, each limited to a small number of sections and none involving the invasive cancer (Fig. 3, Supplementary Fig. S3).In addition to heterogeneity in RNF43 in early lesions, we also identified two cases with multiple mutations in KRAS in precursor lesions, of which only one was present in the invasive cancer.For example, in MTP19, KRAS p.G12V was present in the majority of IPMN samples, as well as all the invasive cancer samples, but there were an additional four other KRAS mutations (all occurring in hotspot positions) that were present in a small number of sections in low-grade IPMN samples (Fig. 3).Intriguingly, these three low-grade IPMN regions shared no mutations with the invasive cancer, suggesting that they represented genetically independent clones.Notably, while there were often many differences in the somatic point mutations identified in the matched noninvasive and invasive samples, the copy number profiles were quite similar between IPMN/MCNs and invasive cancers (Fig. 2, Supplementary Data 6), and the proportion of copy number alterations unique to cancer samples (0.34) was similar to that observed for somatic mutations.While homozygous deletion of some genes occurred in the invasive cancer but not the noninvasive component, such as CDKN2A in MTP8 (Fig. 3), analyses of chromosomal gains and losses through assessment of allelic imbalance revealed that an average of 91% of the genome was similar in copy number in matched noninvasive and invasive samples (Fig. 2, Supplementary Data 6). Insights into pancreatic neoplasia revealed by sequencing.The samples analyzed by targeted sequencing were characterized morphologically and meticulously isolated using laser capture microdissection.Even with this process, we identified samples in two cases that were characterized morphologically as IPMNs but through genomic and evolutionary analyses were determined to be identical to or descendants of the associated invasive cancers.For example, in MTP19, some of the samples originally identified morphologically as noninvasive IPMN (55, 9, 60, and T1) shared all the mutations present in the invasive cancer sample (T2) and contained additional mutations, suggesting that these samples descended from the cancer (Figs.3,4).Evolutionary analyses suggested that some of the morphologically identified IPMN samples in this case (as well as MTP1) actually represented intraductal spread of invasive carcinoma, also referred to as cancerization of the ducts.In these cases, after invading the stroma, the carcinoma invaded back into and colonized the cyst such that it was morphologically indistinguishable from IPMN with high-grade dysplasia. In one case (MTP5), we also identified an interesting pattern of multifocal invasion of the carcinoma.In this case, we analyzed five different samples from invasive cancer-three samples were isolated from a mucinous carcinoma, and two samples were isolated from a ductal carcinoma.Based on evolutionary analyses of the patterns of shared and distinct mutations in the cancers and IPMNs, we conclude that there were multiple separate invasion events in this lesion, as represented by the mutations shared between the invasive cancers and noninvasive components as well as those that were unique to the specific invasive cancers (Fig. 4, Supplementary Fig. S5). As our study represents the largest cohort of comprehensively sequenced IPMNs/MCNs, we also analyzed mutational signatures in our dataset.Intriguingly, our data contrast somewhat with the mutational signatures previously reported in pancreatic ductal adenocarcinoma (PDAC) 16,17 .Like PDAC, the most prominent mutational signature was associated with age (Signature 1A), which was identified in almost every case (Supplementary Fig. S19).However, we also identified signatures associated with APOBEC enzymes (four cases), smoking (three cases), and mismatch repair deficiency (11 cases).Although smoking is considered a risk factor for pancreatic cancer, until now the mutational signature associated with smoking has not been reported in pancreatic neoplasia 18 . Evolutionary timeline of high-grade IPMN to PDAC.To estimate the time between the development of high-grade IPMN and PDAC, we evaluated Bayesian hierarchical models for the number of acquired mutations under a range of possible mutation rates.These models estimate the time interval between a founder cell of a PDAC and the ancestral precursor cell in the associated highgrade IPMN assuming that mutation rates and cell division times are constant throughout this period of development (see \"Methods\").We performed this analysis on the paired WES data from 17 of our 18 cases (Supplementary Fig. S20).We excluded MTP19 because our evolutionary analyses demonstrated intraductal spread of invasive carcinoma and as such, we lacked WES data from an IPMN sample in this case.In the 17 analyzed cases, the average median time to progression from IPMN to PDAC was 3.7 years, but the models showed a bimodal distribution.This median time was nearly 3 years for 13 patients, but nearly 7 years for 4 patients with more than 35 acquired mutations, highlighting potential variability in progression time between patients.For example, in patient MTP1, most models suggested an average of 2.8 years between the development of the IPMN and the PDAC (90% CI, 1.3-6.7 years).In contrast, for patient MTP2 with 36 additional mutations acquired in the PDAC, the transition appears to have been slower with an average estimate of 6.6 years (90% CI: 3.9-11.4years) from the Bayesian models.Overall, these analyses suggest that for most patients there is a significant window of time between development of high-grade dysplasia and pancreatic cancer, providing an opportunity for surveillance and intervention. ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "This study represents the largest dataset of whole exome sequencing of IPMNs and MCNs to date.Importantly, our data established that both IPMNs and MCNs are direct precursors of invasive pancreatic cancer (Fig. 4).This conclusion has been previously suggested by the morphological relationship between the noninvasive neoplasms and invasive cancer on traditional histologic sections, as well as shared driver gene mutations in targeted sequencing studies 6,7,[9][10][11] .However, the presence of many shared driver and passenger mutations clearly demonstrated the common origin of IPMNs/MCNs and invasive pancreatic cancers in our study, and evolutionary analyses revealed that dysplastic lesions precede invasive cancers.Evolutionary analyses suggested that high-grade noninvasive lesions occur over 3 years before invasive carcinoma, providing a window of opportunity for early detection and intervention. In this study, we identified somatic mutations in driver genes that had not been previously implicated in IPMNs/MCNs.For example, we identified alterations in the DNA damage response gene ATM in 17% of the analyzed cases.Germline mutations in ATM have been recently reported in patients that developed IPMNs, highlighting the potential importance of this gene in IPMN risk 19 .In addition, mucinous (colloid) carcinomas are significantly more common than typical ductal carcinomas in patients with germline ATM mutations, further highlighting the link between mutations in this gene and IPMNs 20 .Although the ATM gene is large, potentially increasing the likelihood of passenger or artifactual mutations, even larger genes such as TTN had a lower mutation prevalence, suggesting that at least some of the alterations identified in ATM are likely to be bona fide somatic mutations.Somatic mutations in GLI3, which encodes a component of the Hedgehog signaling pathway, were identified in 28% of cases.Somatic mutations in GLI3 were recently reported in a distinct morphological variant of pancreatic carcinoma (undifferentiated carcinoma with osteoclast-like giant cells) as well as at a low prevalence in sporadic PDAC, suggesting that the importance of GLI3 mutations and its signaling pathway in pancreatic tumorigenesis may extend beyond IPMNs/MCNs [21][22][23] .The hotspot mutations in SF3B1, which encodes a protein critical for RNA splicing, are also potential drivers in the IPMN pathway.However, somatic mutations in this gene have been reported in a variety of other neoplasms, including hematologic malignancies and uveal melanoma [24][25][26] . We highlight somatic alteration of the SMAD4 pathway as a putative driver of progression to invasive cancer, as mutations in SMAD4 or TGFBR2 occurred only in invasive cancer samples in 4 of the 18 cases analyzed.SMAD4 was the only gene with cancerspecific mutations in more than one case, highlighting the potentially unique role this gene plays in pancreatic carcinogenesis.This role has been previously suggested by next generation sequencing of high-grade PanINs showing an absence of SMAD4 mutations in precancerous lesions, as well as cancer-specific SMAD4 mutations reported in a paired PanIN/carcinoma analyses 27,28 .Loss of SMAD4 expression limited to invasive carcinomas has been reported in MCN-and IPMN-associated invasive cancers, and targeted sequencing of a small number of IPMNs and matched cancers identified a single case with a SMAD4 mutation occurring only in the cancer 7,28,29 .In our data, there were also four cases where mutations in SMAD4 were shared between noninvasive and invasive cancer samples, and two where SMAD4 mutations were limited to the noninvasive component.Although our whole-exome approach could not detect all types of SMAD4 alterations (such as rearrangements or epigenetic changes), the majority of SMAD4 mutations observed in both noninvasive and invasive components affected both alleles.Taken together, this suggests that the role of SMAD4 mutations may not be universal and may depend on other factors, including cell intrinsic (such as somatic mutations in other driver genes) and cell extrinsic (such as stromal and immune microenvironment) mechanisms. Although some of our cases had SMAD4 mutations limited to the invasive cancer, most of the IPMN/MCN-associated cancers lacked driver gene alterations that were associated with invasive disease, suggesting that malignant progression is not universally driven by point mutations.Previous studies have specifically demonstrated the importance of copy number alterations and chromosomal rearrangements in pancreatic tumorigenesis 30 .We did not identify large differences in the copy number profiles between noninvasive components and associated invasive cancers, suggesting that global genomic instability may be important as an early feature of tumorigenesis but is not likely to drive malignant transformation in many cases. Our study also revealed prevalent genetic heterogeneity in driver gene mutations in early lesions, demonstrating more complex processes than previously suggested by traditional linear tumorigenesis models.Similar to our recently reported polyclonal origin of IPMNs 12 , we identified multiple independent clones initiated by distinct KRAS mutations in two cases in the current study.In addition, our study identified multiple distinct inactivating mutations in RNF43 limited to unique tumor subclones, a pattern previously observed by our group and not shared by other genes in our whole exome sequencing analyses 12,31 .In most cases, RNF43 mutations were enriched in noninvasive components and absent from the associated invasive cancers.More generally, we observed multiple instances of clear driver mutations (including hotspot mutations in oncogenes as well as inactivating mutations in tumor suppressor genes) that were limited to the noninvasive components and not present in the associated invasive cancer.Thus, these mutations occur and clonally expand in the IPMN or MCN but are not present in the subclone that subsequently invades.Such independent evolution of premalignant lesions has been observed in other organ sites and does not diminish the conclusion of our evolutionary analyses that IPMNs/MCNs are precursors of invasive pancreatic cancer [32][33][34] .Rather, these observations suggest unique selective processes at different time points in tumorigenesis, such that mutations selected in the precancerous lesion are not selected for (or are even selected against) in the invasive cancer. In addition to these observations about clonal evolution in noninvasive lesions, our data also provide genetic evidence for multiple underappreciated processes in pancreatic neoplasia.First, we provide genetic evidence for intraductal spread of invasive carcinoma, also known as cancerization 35 .In two of our cases (MTP1 and MTP19), the identified somatic mutations suggested that samples that were morphologically thought to be IPMN were actually of the same clone or clone descended from invasive cancer.These cases confirmed the morphological impression of the prevalence of this cancerization phenomenon, which likely has confounded many previous studies of precancerous pancreatic lesions 35 .In addition, we describe one case of IPMN with multiple independent invasion events (MTP5).This case contained invasive cancer with two different morphologies, one with typical ductal morphology and one with mucinous (colloid) morphology.Evolutionary analyses demonstrated that the ductal and mucinous carcinomas arose through independent invasion events and suggested that the multiple mucinous cancer samples comprised unique subclones that invaded independently from the IPMN.Although multifocal invasion has been described morphologically in IPMNs with multiple anatomically discrete invasive foci, in this case all the invasive carcinoma samples came from the same grossly defined tumor, suggesting that multifocal invasion may be an underappreciated phenomenon in IPMNs. The results of our study should also be considered in the context of studies of other precancerous lesions and associated invasive cancers.Previous molecular studies of IPMNs and associated invasive carcinomas have been limited to targeted gene panels [9][10][11] .These studies demonstrated a small number of shared and distinct driver gene mutations in lesions from the same patient.The results of these studies are largely consistent with our study, but the limited panels prevent comprehensive evolutionary conclusions.In addition, complementary studies that have employed whole exome sequencing to characterize microscopic pancreatic precancerous lesions (PanINs) and their co-occurring invasive carcinomas highlight the common evolutionary origin of PanINs and co-occurring PDACs 27,36 .Similar to our study, these studies reported a lack of consistent specific driver genes associated with invasive cancer, although cancer-specific SMAD4 mutations were reported in two cases in one study 27 .Importantly, in our study the sequencing of additional precancerous samples (beyond the original paired samples analyzed by whole exome sequencing) provided a more detailed analysis of precancerous clonal evolution than previous studies. As with all genomic analyses, our study does have some limitations.Compared to other genomic analyses of invasive pancreatic cancer, the sample size in our study is relatively small, with multi-region whole exome sequencing performed on 18 patients.Nevertheless, the whole exome and targeted analyses of 148 samples from these patients represents the largest genomic study of precancerous pancreatic lesions to date.In addition, our combined approach of whole exome and targeted sequencing may not have identified all mutations in all regions of these comprehensively analyzed IPMNs.Despite these limitations, the analyses provide a detailed view of the acquisition of mutations that characterize the invasive carcinoma, as well as genetic heterogeneity in well-characterized pancreatic driver genes.Finally, in this study, we focused entirely on genetic alterations, as the role of these mutations in driving tumorigenesis has been well documented.Our study provides evidence that the evolution to invasive cancer is likely to be driven by non-genetic mechanisms in some lesions, highlighting an important direction of future investigation. In this study, we present a comprehensive evolutionary analysis of precancerous pancreatic cysts and associated invasive carcinomas.We demonstrate that IPMNs and MCNs are precursors of invasive pancreatic cancer, that alterations in ATM, GLI3, and SF3B1 are present in these lesions, and that SMAD4/TGFBR2 alterations are likely drivers of invasion in a subset of cases.Analyses of the evolutionary timeline between high grade precancerous lesions and pancreatic cancer suggest a window of more than 3 years for acquisition of these invasive characteristics.These data provide critical insights into pancreatic tumorigenesis and highlight an opportunity for surveillance of precancerous pancreatic cysts and early detection of pancreatic cancer. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Specimen acquisition.An IRB protocol was submitted and approved at the Johns Hopkins University.Additional IRB protocols were submitted and approved in collaborating institutions when a local IRB was required.Samples came from the Johns Hopkins Hospital, Baltimore (United States); University Medical Center, Utrecht (The Netherlands); University and Hospital Trust, Verona (Italy); Asan Medical Center, Seoul (Republic of Korea); National Cancer Center Hospital, Tokyo (Japan); Royal North Shore Hospital, Sydney (Australia), University Hospital, Ghent (Belgium); Academic Medical Center, Amsterdam (The Netherlands); Laboratory for Pathology Eastern Netherlands, Hengelo (The Netherlands); Thomas Jefferson University, Philadelphia (United States); Aichi Cancer Center Hospital, Nagoya (Japan); Medica Sur Clinic and Foundation, Mexico City (Mexico); Emory University Hospital, Atlanta (United States).Informed consent was obtained from all participants in accordance with IRB requirements.The pathology archives were searched for cases meeting the following inclusion criteria: synchronous IPMN or MCN and pancreatic cancer, whereby the IPMN or MCN is larger than the invasive component and the invasive component is smaller than 2.5 cm.We specifically sought small pancreatic cancers to isolate the alterations that occur at the time of malignant progression (rather than those that accumulate during further growth of the invasive cancer).The H&E slides were evaluated by experienced pathologists with a subspecialty in gastrointestinal pathology (L.D.W., R.H.H., L.A.A.B., G.J.O.), and the different components (noninvasive cyst and invasive cancer) were annotated. Sample preparation and whole exome sequencing.The formalin-fixed paraffinembedded (FFPE) tissue blocks were cored separately for the different components: high-grade IPMN/MCN, invasive cancer, and matched normal sample from the duodenum or spleen.As these are human tissue blocks from pathology archives of the participating hospitals, these materials are not available to the public.In three cases, a low-grade IPMN/MCN was also cored.DNA was extracted from these cases using the Qiagen QIAamp DNA FFPE Tissue Kit according the manufacturer's protocol (Qiagen).After purification, the final DNA concentration was measured with the Qubit 2.0, dsDNA high sensitivity assay.Noninvasive lesions invariably had a higher neoplastic cellularity, as assessed during pathology review.Twenty cases were selected for whole-exome sequencing (WES) of the noninvasive component(s), cancer, and matched normal at Personal Genomic Diagnostics (PGDx, Baltimore).Briefly, genomic DNA was fragmented, followed by end-repair, A-tailing, adapter ligation, and polymerase chain reaction.PCR products were purified, and exonic regions were captured in solution using the Agilent Sure Select kit according to the manufacturer's instructions (Agilent).Pair-end sequencing was performed on a Hi-Seq2500 next-generation sequencing instrument (Illumina).Primary processing of sequence data was performed using Illumina CASAVA software (v1.8).Two cases (MTP15 and MTP20) failed quality control during sequencing and were excluded from further analysis.Fastq-files were further processed according to the GATK best practices workflow: BWA (v0.7.17) for alignment to hg19, picard (v2.18.1) for duplicate reads flagging and GATK (v3.7) for base quality score recalibration. Somatic mutation identification from whole exome sequencing data.Nonsynonymous mutations were called with PGDx's VariantDX mutation caller, and synonymous mutation were called using Mutect2 (in GATK v3.7), both using default parameters 37,38 .All identified mutations were confirmed by visual inspection using the Integrated Genomics Viewer (v2.3.80;IGV) 39 .In order to assess mutated sites in additional samples from the same patient, a separate analysis was performed with Manta (v1.4.0) and Strelka (v2.9.9) 40 .To obtain information on the mutated positions across all samples from each patient, Strelka was used to obtain metrics at each mutated position in all matched samples.Then, mutations were filtered based on the several metrics.A mutation was called when the normal sample had a depth of at least 10 distinct reads and the mutation found had a mutant allele fraction of <2% in the normal.Further filtering is described in \"Integrated mutation analysis\" below.In addition, analysis of each candidate mutation was performed using BLAT (v36) 41 .A sequence of 101 bases (with the mutated position in the middle) was used as the query sequence (http://genome.ucsc.edu/cgi-bin/hgBlat). Candidate mutations were removed from further analysis if the analyzed region resulted in >1 BLAT hits with 90% identity over 70 SCORE sequence length.All candidate alterations were verified by visual inspection in IGV. In order to analyze the mutational signatures, we combined the single nucleotide variants found in both the noninvasive sample(s) and the invasive sample into a set of unique mutations.Each mutation was classified into one of the 96 trinucleotide contexts 18 .The contribution of each signature to each tumor sample was estimated using the deconstructSigs (v1.8.0) R package. Sample preparation and targeted sequencing.Additional blocks were available from seven of the analyzed IPMNs/MCNs, allowing multi-region sampling of both the precursor lesion and the pancreatic cancer.Twenty to thirty serial sections of FFPE tissue from each available block were cut onto membrane slides (PEN 1.0, Zeiss, Oberkochen, Germany) for laser capture microdissection.After deparaffinization in xylene and rehydration in ethanol, the slides were stained with hematoxylin. Morphologically homogeneous regions were microdissected using the LMD7000 (Leica, Wetzlar, Germany).Genomic DNA was extracted from the microdissected tissues.In brief, the microdissected samples were incubated with proteinase K for 16 h at 56 °C.The digested mixture was transferred to a 130 μL microtube (Covaris, Woburn, MA) for shearing.Following fragmentation, the sample was further digested for 24 h at 56 °C followed by 1 h incubation at 80 °C to inactivate proteinase K. DNA purification was then performed using the QIAamp DNA FFPE Tissue kit following the manufacturer's instructions (Qiagen, Valencia, CA).Fragmented genomic DNA from tumor and normal samples were used for library preparation according to the Agilent Sure Select Target Enrichment System (Agilent, Santa Clara, CA).The targeted panel (Agilent) was constructed, containing all the regions with the mutations identified in the WES from these seven cases.The protein coding regions of 15 pancreatic cancer driver genes (APC, ATM, BRAF, CDKN2A, CTNNB1, GNAS, KRAS, MAP2K4, PIK3CA, PTEN, RNF43, SMAD4, STK11, TGFBR2, TP53) were also included in the targeted panel 12 .Library preparation was performed using the Agilent Sure SelectXT Target Enrichment System (Agilent) following the manufacturer's instructions with the following modifications: genomic DNA was sheared prior to library preparation (see above).In addition, half of the volume of capture library was used per reaction, and supplemented with water.Finally, both pre-and post-capture PCR cycle number was increased by 0 or 1 for DNA inputs of 200 ng or 50-100 ng, respectively.Paired-end sequencing was performed using the Illumina MiSeq (Illumina, San Diego, CA). Somatic mutation identification from targeted sequencing data.Samples with an average sequencing depth of >150× in the targeted regions were included for further analysis.Mutations were identified by Strelka, specifying the union of mutations found in the samples of the each patient as input for a second round of analysis 40 .This approach yielded mutation metrics (reference and alternate read counts) across all samples from each patient.Only mutations identified in the whole exome data of each patient or mutations found in the 15 driver genes that were fully sequenced were considered for further analysis.Newly discovered mutations in the fully sequenced genes were visually inspected in IGV. ",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "The following criteria were applied to establish presence or absence of mutations in each analyzed sample profiled by whole exome or targeted sequencing, which subsequently informed the phylogenetic analysis.To start, the median depth of coverage over the capture region was calculated for each sample.The mutated loci selected for analysis in each patient were required to have a sequencing depth of coverage exceeding 20% of the median coverage of the sample, in all sequenced samples of the patient.This strategy enabled exclusion of loci where insufficient or inconsistent capture efficiency might restrict the power to detect mutations.In a whole exome sequenced samples, a minimum mutant allele fraction of 10% and five mutant reads was used to define mutation presence (primary mutation calls).However, our analysis of tumor purity revealed a marked difference between the level of normal contamination in precursor and cancer lesions.Given that reduced tumor purity results in lower levels of mutant allele fraction and mutant read counts, for mutations marked as present in at least one sample of each patient, a minimum of two mutant read counts was deemed sufficient to call the mutation present in a whole exome sequenced samples (secondary mutation calls).In samples analyzed by targeted sequencing, a minimum mutant allele fraction of 5% and a sample-specific minimum mutant read count were applied to mark a mutation as present.The lowered mutant allele fraction was applied to take advantage of the fact that the higher depth of coverage in targeted sequencing can allow sensitive mutation detection at lower MAF levels without compromising accuracy.The sample-specific minimum mutant read count was obtained by scaling the value 5 (threshold used for whole exome sequenced sample), by the ratio of the median coverage of the targeted sample to the average median coverage of the whole exome samples of that patient.Increasing the minimum required value for the mutant read count helped avoid false positive calls due to sequencing errors at increasingly high levels of coverage. Copy number evaluation.In samples analyzed by whole exome sequencing, we applied FACETS (v0.5.14) to determine the tumor purity and ploidy of the sample, as well as allele-specific copy number for regions across the genome 42 .In each tumor sample, we investigated focal copy number aberrations (focal CNAs) by focusing on genomic regions with length <3 Mb where the estimated copy number was at zero (homozygous deletion), or was greater than or equal to three times the estimated ploidy of the sample (focal amplifications).We filtered the focal copy number aberrations to those that passed the visual review in the following categories: (1) In samples with tumor purity of 30% or above, any aberrations affecting the 15 driver genes frequently mutated in pancreatic cancer regardless of their estimated cancer cell fraction were reviewed.In addition, we reviewed other focal CNAs with cancer cell fraction of 75% or above (likely clonal).( 2) In samples with tumor purity below 30%, we reviewed all changes affecting an in-house set of 195 cancer driver genes if their estimated cancer cell fraction was 75% or above.Finally, we visually inspected each detected focal CNA in the other whole exome sequenced samples from the same individual to enable recovery of potentially false negative calls due to technical issues.This step allowed us to use focal CNAs as an additional class of features in phylogenetic analysis. In whole exome sequenced samples, the allele specific copy number values estimated by FACETs were used to determine whether a locus harboring a somatic mutation has undergone LOH, as indicated by minor copy number of zero.In seven cases where a number of lesions were profiled by targeted sequencing, the small size of the panel (~185 kb) prohibited analysis by conventional tools.Therefore, we applied a custom analysis pipeline to evaluate the copy number status of the 15 driver genes whose coding sequence was fully covered by the panel as follows.First, the base level depth of coverage for all positions on the panel were calculated and summarized by taking the median across each interval on the panel.Next, these values were then normalized by the median of the coverage for all the intervals on the panel, and corrected for GC content.The resulting copy ratio values were aggregated for each driver gene by taking the median of all corresponding genomic intervals.Finally, homozygous deletion was defined as a copy ratio value below -1.25 in log2 scale (using a 0.25 margin of error to allow differentiation from hemizygous deletion), and focal amplification was defined as a copy ratio value exceeding 1.6 in log2 scale (corresponding to copy number 6 or above in a largely diploid genome). Genome-wide assessment of allelic imbalance and LOH.A comparative analysis of LOH across the tumor samples of each patient was performed to identify structural alterations occurring in the course of tumor evolution as described previously 32 .Given the difference in the breadth of genome coverage in whole exome and targeted sequenced samples, the number of informative loci (germline heterozygous SNPs) widely varies between the two approaches.Therefore, our analysis started by evaluation of LOH in whole exome samples and was later extended to targeted samples in regions were SNPs with sufficient coverage were available in targeted sequencing data.Circular binary segmentation was applied to the minor allele frequency (mAF) of germline heterozygous SNPs in each whole exome sequenced sample to determine genomic region with a constant level of allelic imbalance 43 .In each sample, a difference of 0.1 between the segment mAF of tumor and matched normal samples was required to label a region as harboring LOH.Across genomic segments with lengths exceeding 10 Mb and overlapping at least 20 SNPs, the minimum of segmental mAF was recorded and used as proxy measure reflecting the purity of the tumor sample.The confidence in LOH calls for each segment was determined by comparison of the segment mAF with the minimum sample-level mAF and reported in three tiers: (1) high confidence tier: segment mAF is within 0.1 distance of minimum sample-level (2) intermediate confidence tier: segment mAF distance to minimum sample-level maf is in 0.1-0.2range (3) low confidence tier: segment mAF exceeds the minimum sample-level mAF by at least 0.2.Next, the union of the genomic coordinates for segment breakpoints across all whole exome sequenced samples of each patient was derived and the segmental mAF and the number informative SNPs in each sample was calculated in the intervals defined by each pair of consecutive breakpoints (patient-level segments).The resulting segments were filtered to those spanning a minimum of 10 Mb, overlapping 20 informative SNPs, and belonging to the high confidence LOH tier in at least one sample of the patient.The segments passing the above filters were categorized into two sets: those uniformly present across all, and those differentially present in a subset of whole exome sequenced samples.To increase the specificity, the latter set was further narrowed down to the set where there was a minimum difference of 0.1 between the segmental mAF of samples with and without LOH.To avoid overestimation of the number of independent structural alterations, LOH segments with boundaries within a 5 Mb window and identical LOH calls across all samples analyzed were merged together. This analysis was extended to include targeted sequenced samples as follows.First, the number of reference and alternate alleles at each germline heterozygous SNP were determined and the SNPs were filtered to those with minimum distinct coverage of 20×.For each segment, the count and average mAF of the overlapping SNPs were calculated.LOH of each candidate segment from whole exome analysis was determined using the criteria described above, and segments with LOH were classified into the three confidence tiers.This approach yielded a candidate set of LOH changes which can be used in conjunction with somatic mutations to inform the phylogenetic analysis.Moreover, the LOH status across the genome can be used to annotate mutations in each sample, and highlight cases where the absence of mutation in a sample is due to structural alterations as opposed to intra-tumoral heterogeneity across the samples.Phylogenetic analysis.To derive a parsimonious description of tumor evolution in each patient, the set of somatic alterations (mutations, LOH changes, focal CNAs) identified was first represented as binary matrix reflecting their presence in each analyzed sample.The unique patterns of presence/absence observed across samples of each patient were determined and used to define alteration clusters.These clusters comprise of the entire set of alterations with a common pattern of presence across the samples.To avoid spurious assignment of singular alterations to individual clusters due to false positive or negative calls, the initial set of clusters were filtered to those with at least two alterations.The remaining set of alterations belonging to singleton clusters were reviewed to determine whether they can be merged with any of the existing clusters, by considering the quality of mutation or LOH calls in samples where they differed; i.e., the distance of mutation/LOH change metrics from the thresholds used to establish the calls and evaluating the possibility of mutation loss due to LOH of the mutated loci and copy number loss.At this point, the presence pattern of each cluster was assumed to represent that of all its constituent mutations and was used to correct any erroneous calls identified through the review process.In 13 patients where all samples were whole exome sequenced and allele-specific copy number was uniformly available across all the samples, mutations where the absence in subset of samples could be explained by either copy number loss or intra-tumoral heterogeneity were excluded from phylogenetic analysis to avoid ambiguity. Given the focus of the current study on evaluation of heterogeneity and evolution among lesions in each patient and not the clonal heterogeneity within each lesion, the genotype of cancer cells within each lesion was assumed to be uniform and defined by the presence of alterations identified.However, in cases where a subset of samples are defined by co-existence of two or more clonal populations, such an assumption will result in inaccurate phylogenetic reconstruction on the level of samples as the mixed samples cannot be represented by a unique genotype.Therefore, we sought to identify cases with possible clonal mixing and applied a correction method inspired by one of conceptual framework of SCHISM (v1.1.2) as follows 44 .First we evaluated all pairs of mutation clusters and identified the subset where neither of the two members of the pair can be ancestral to the other in the phylogenetic tree; i.e., the set of samples harboring each member of the pair are not nested within each other.Such pairs indicate mutation clusters acquired on distinct branches of the phylogenetic tree, and should not co-occur in any tumor samples in absence of clonal mixing.The set of mutation cluster pairs (a i , b i ) is narrowed down such that no potential ancestor of a i is present with b i as a pair in the set and vice versa.Next, the set is further filtered down to mutation cluster pairs that do co-occur with each other in at least one tumor sample.Finally, any tumor sample harboring both members of such a mutation cluster pair is represented as two distinct populations of cancer cells (subclones) each defined by the presence of one member of the pair and the absence of the other and its potential descendants (mutation clusters present in a subset of samples where the original cluster is present). The binary matrix resulting from the analysis above were used as inputs to the maximum parsimony phylogeny module (pars) in PHYLIP (v3.695, http:// evolution.genetics.washington.edu/phylip/), and the resulting phylogenetic trees were visualized using the ggtree (v1.4.11) module in R. Analysis of timing of malignant progression.Our sampling model for the number of additional mutations acquired in PDAC sample i is Poisson with mean θ i .The average number of mutations θ i can be factored as the product of the mutation rate μ (assumed to be the same for all patients) and the number of years T i during which the y i mutations accumulated.The second stage of the model posits a Gamma sampling distribution for the timing between the birth of the highgrade IPMN and the PDAC.Finally, we use diffuse priors for the shape and rate parameters of the Gamma.The model implemented in JAGS version 4.3.0 is 2s 2 m $ Uniformð0; 100Þ s $ Uniformð0; 100Þ; where m and s correspond to the mode and standard deviation of the gamma prior for T. As the mutation rate μ is not known, we implemented this model for a range of plausible values (1 mutation/year-10 mutations/year). Statistics and reproducibility.All neoplastic samples were sequenced once: through whole exome sequencing or through targeted sequencing.In cases with both whole exome and targeted sequenced, normal samples were sequenced both with the whole exome probes and the targeted probes.Otherwise, normal samples were sequenced once. ",
"section_name": "Integrated mutation analysis.",
"section_num": null
}
] |
[
{
"section_content": "The authors acknowledge the following sources of support: NIH/NCI P50 CA62924; NIH/NIDDK K08 DK107781; NIH/NCI R01 CA121113; NIH/NCI R00 CA190889; NIH/ NCI P30 CA006973; Sol Goldman Pancreatic Cancer Research Center; Buffone Family Gastrointestinal Cancer Research Fund; Carol S. and Robert M. Long Pancreatic Cancer Research Fund; Kaya Tuncer Career Development Award in Gastrointestinal Cancer Prevention; AGA-Bernard Lee Schwartz Foundation Research Scholar Award in Pancreatic Cancer; Sidney Kimmel Foundation for Cancer Research Kimmel Scholar Award; AACR-Incyte Corporation Career Development Award for Pancreatic Cancer Research; American Cancer Society Research Scholar Grant; Emerson Collective Cancer Research Fund; Rolfe Pancreatic Cancer Foundation; Joseph C Monastra Foundation; The Gerald O Mann Charitable Foundation (Harriet and Allan Wulfstat, Trustees); Susan Wojcicki and Denis Troper; Dr. Miriam and Sheldon G. Adelson Medical Research Foundation; Dutch Digestive Foundation (MLDS CDG 14-02); Nijbakker-Morra Foundation; Lisa Waller Hayes Foundation; Avner Pancreatic Cancer Foundation. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "When permitted by the relevant IRBs (77 samples), whole exome and targeted sequencing data has been deposited in the European Genome-phenome Archive with accession EGAS00001004473. Code for the analysis of the timing of malignant progression is available at https://gitlab.com/cancer-genomx/ipmn-timing. ",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "When permitted by the relevant IRBs (77 samples), whole exome and targeted sequencing data has been deposited in the European Genome-phenome Archive with accession EGAS00001004473. ",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "Code for the analysis of the timing of malignant progression is available at https://gitlab.com/cancer-genomx/ipmn-timing. ",
"section_name": "Code availability",
"section_num": null
},
{
"section_content": "M.N., L.A.A.B., and L.D.W. designed the study.M.N., W.H., C.L., A.P., A.S., G.B., G.Z., P.C., S.M.H., S.Y., N.H., A.J.G., J.S.S., G.J.A.O., A.H., J.V., C.J., N.V.A., W.J., J.W., J.A.S., B.T., E.D.T., L.A.A.B., and L.D.W. analyzed tissue samples.M.N., C.G.F., W.M.H., W.H., M.D., E.P., V.A., L.A.A.B. acquired data.M.N., N.N., V.B.G., J.R.W., N.J.R., R.K., R.B.S., and L.A.A.B. analyzed data.M.N., N.N., R.H.H., R.K., R.B.S., L.A.A.B., V.E.V., and L.D. W. interpreted data.M.N., V.E.V., and L.D.W. wrote the paper.All authors have approved the submitted version of the paper. L.D.W. receives research funding from Applied Materials.V.E.V. is a founder of Personal Genome Diagnostics, a member of its Scientific Advisory Board and Board of Directors, and owns Personal Genome Diagnostics stock, which are subject to certain restrictions under university policy.V.E.V. is an advisor to Takeda Pharmaceuticals.Within the last five years, V.E.V. has been an advisor to Daiichi Sankyo, Janssen Diagnostics, and Ignyta.J.R.W. is founder and owner of Resphera Biosciences LLC, and is a consultant to Personal Genome Diagnostics Inc.The terms of these arrangements are managed by Johns Hopkins University in accordance with its conflict of interest policies.The other authors declare no conflict of interest. Supplementary information is available for this paper at https://doi.org/10.1038/s41467-020-17917-8. Correspondence and requests for materials should be addressed to L.D.W. Peer review information Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work.Peer reviewer reports are available. Reprints and permission information is available at http://www.nature.com/reprintsPublisher'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": "M.N., L.A.A.B., and L.D.W. designed the study.M.N., W.H., C.L., A.P., A.S., G.B., G.Z., P.C., S.M.H., S.Y., N.H., A.J.G., J.S.S., G.J.A.O., A.H., J.V., C.J., N.V.A., W.J., J.W., J.A.S., B.T., E.D.T., L.A.A.B., and L.D.W. analyzed tissue samples.M.N., C.G.F., W.M.H., W.H., M.D., E.P., V.A., L.A.A.B. acquired data.M.N., N.N., V.B.G., J.R.W., N.J.R., R.K., R.B.S., and L.A.A.B. analyzed data.M.N., N.N., R.H.H., R.K., R.B.S., L.A.A.B., V.E.V., and L.D. W. interpreted data.M.N., V.E.V., and L.D.W. wrote the paper.All authors have approved the submitted version of the paper. ",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "L.D.W. receives research funding from Applied Materials.V.E.V. is a founder of Personal Genome Diagnostics, a member of its Scientific Advisory Board and Board of Directors, and owns Personal Genome Diagnostics stock, which are subject to certain restrictions under university policy.V.E.V. is an advisor to Takeda Pharmaceuticals.Within the last five years, V.E.V. has been an advisor to Daiichi Sankyo, Janssen Diagnostics, and Ignyta.J.R.W. is founder and owner of Resphera Biosciences LLC, and is a consultant to Personal Genome Diagnostics Inc.The terms of these arrangements are managed by Johns Hopkins University in accordance with its conflict of interest policies.The other authors declare no conflict of interest. ",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Supplementary information is available for this paper at https://doi.org/10.1038/s41467-020-17917-8. Correspondence and requests for materials should be addressed to L.D.W. Peer review information Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work.Peer reviewer reports are available. Reprints and permission information is available at http://www.nature.com/reprintsPublisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ",
"section_name": "Additional information",
"section_num": null
}
] |
10.26444/aaem/106041
|
Rare case of Richter’s syndrome localization in liver and thyroid of a patient with a chronic lymphocytic leukemia (CLL) – Case report and literature
|
Richter's syndrome (RS) is a rare complication in which chronic lymphocytic leukaemia (CLL) or small lymphocytic lymphoma (SLL) transforms into a more aggressive type of lymphoma - diffuse large B cell lymphoma (DLBCL), or Hodgkin's lymphoma (HL). The review describes the clinical case of a patient with CLL and RS diagnosis. A computed tomography (CT) scan of the abdominal cavity detected numerous normodense areas in the liver. Simultaneously, ultrasound examination (USG) of the thyroid revealed the presence of a solid hypoechogenic lump. The material sampled from closed biopsies of liver and thyroid in both cases allowed the diagnosis of diffuse large B cell lymphoma (DLBCL). The liver and the thyroid are particularly rare locations of RS. However, those cases allowed the conclusion that RS may occur even in a very unexpected and less probable location.
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is usually a lymphoproliferative disease with a chronic course [1,2,3].It is the most commonly diagnosed type of leukemia in adults.The disease is characterized by the variety of clinical course, prognosis and response to cytostatic treatment; the start of treatment is individual for each patient and depends on the severity of the clinical or progressive nature of the disease.In some patients, mild course and long survival are observed, very often without cytostatic treatment.In the remaining patients, the disease is aggressive and despite intense therapy, it leads to death within 2-3 years. Richter's syndrome is defined by the transformation of chronic proliferation of B-cells, which is chronic lymphocytic leukemia or lymphocytic lymphoma, into a more aggressive form of lymphoma.Most commonly, it is a diffuse large B-cell lymphoma (DLBCL-RS) [4], rarely Hodgkin lymphoma (Hodgkin variant of RS, HvRS) [5].The frequency of occurring of RS among patients with CLL is estimated to be 2%-10% [6] The most frequent RS (80%-90%) stems from the same clone as CLL (clonally related RS).In a very small part, the tumor clone develops independently (clonally unrelated RS) [7].The increased risk of RS occurring is related with the presence of genetic aberrations, such as del 11q and 17p [8,9], unmutated IGHV, significant expression of ZAP-70, CD38 and CD49d [10,11].RS is characterized by very rapid clinical process.Among more than half of patients (about 59%) there is fever, weight loss or intensified, night hyperhidrosis [12,13].RS most commonly occurs in lymph nodes and bone marrow.Localization in the lymphatic system occurs in about 41% of cases.Particularly rare cases are those, where transformations occur in the liver and thyroid [14,3]. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "In June 2007, 60-year-old woman, on the basis of histopathological examination of lymph node and immunophenotype of peripheral blood lymphocytes (CD19+/CD5+/CD23+; Ig lambda+), was diagnosed with chronic lymphocytic leukemia (stage 4 according to Rai classification).Complete peripheral blood count revealed: WBC 24.8 K/uL; Lymph.21.0 K/uL; Hgb 9.9 g/dL; PLT 70.0 K/ul, concentration LDH 132.0 U/l (10-480).The patient had not reported any general symptoms.Leukemia cells indicated immunological phenotype ZAP70-/CD38+, without the presence of negative genetic aberrations, such as del 11q and del 17p.Physical examination revealed peripheral lymphadenopathy, and USG examination revealed increased size of lymph nodes in the abdominal cavity, without signs of organomegaly.Treatment was started (totally, 6 cycles with chlorambucil and prednisone), resulting in complete remission (CR) of the underlying disease.Afterwards, the patient was observed on an outpatient basis. In February 2015, the patient was admitted to a hospital due to gradually increasing upper abdominal pain, anorexia and weight loss which had lasted for several weeks.Complete peripheral blood count revealed: WBC 9.79 K/uL, Lymph.6.75 K/uL, Hgb 11.2 g/dL, PLT 133.0 K/ uL and LDH 753.0 U/l.CT examination revealed also an increased liver size (200 mm in midclavicular line), with irregular outline, displacing neighbouring organs.Reduced vascular pattern, organ parenchyma with density about 40 u.H., almost the whole covered by numerous, segmentally confluent, round normodense areas, showing minor contrast enhancement.Areas of a similar character had been also observed on an increased in size (177mm) spleen (Fig. 1).Furthermore, there were observed numerous, increased in size, lymph nodes located in retroperitoneum.Leukemia lymphocytes indicated phenotype ZAP70-/CD38+ with increased, compared to the time of CLL diagnosis, share of CD38+ cells (36% vs 49%), again without presence of genetic aberrations.Biochemical examinations also revealed increased activity of transaminases: Alat 104.0 U/l (5-45) and Aspat 161.0 U/l (5-50).Patient also had closed core biopsy of the liver and a sample of the material was taken for histopathological examination.The biopsy revealed a large lymphoid cells infiltration with morphology of centroblasts and immunoblasts (CD20+; bcl6+; CD10-; bcl2-; CD3-; CD5-; cyclin D1-; Ig lambda+; Ki67 about 90%).Results of immunohistochemical examinations led to diagnose the transformation of CLL into diffuse large B-cell lymphoma -Richter's syndrome (Fig. 2). At the same time, CT of the chest suggested increased size of thyroid with presence of subcarinal set of lymph nodes (sized 40 x 30mm).USG examinations of the thyroid revealed the presence of a hypoechogenic lump in the left thyroid lobe and isthmus of the thyroid gland.Concentration of hormones was equal to: TSH 1.67 uIU/ml (0.27-4.2);FT4 0.95 ng/dl (9.0-20.0);FT3 2.57 pg/ml (4.0-8.3).In the course of the performed fine-needle biopsy, a sample of thyroid and nearby lymph nodes were taken for histopathological examination.Immunohistochemical reactions of the large lymphoid cells infiltration presented the following expression: CD20+, bcl6+, CD10-, bcl2-, CD5-, CD3-, Ki67 about 90%.The final diagnosis indicated diffuse large B-cell lymphoma in both samples (Fig. 3). From March -November 2015, the patient received 8 cycles of R-CHOP immunochemotherapy.In December, after performing evaluation of clinical condition of the patient (CT scan, punch biopsy), complete remission was observed of the underlying disease.The patient is still under clinical observation. ",
"section_name": "CASE REPORT",
"section_num": null
},
{
"section_content": "Richter's syndrome is defined by transformation of chronic proliferation of B-cells, which is chronic lymphocytic leukemia or lymphocytic lymphoma, into a more aggressive form of lymphoma.Most commonly, it is a diffuse large B-cell lymphoma (DLBCL-RS) [4], rarely Hodgkin lymphoma (Hodgkin variant of RS, HvRS) [5].The frequency of occurrence of RS among patients with CLL is estimated to be 2%-10% [6] The most frequent RS (80%-90%) stems from the same clone as CLL (clonally related RS).In a very small part, a tumour clone develops independently (clonally unrelated RS) [7].The increased risk of RS occurring is related with presence of genetic aberrations, such as del 11q and 17p [8,9], unmutated IGHV, significant expression of ZAP-70, CD38 and CD49d [10,11].RS is characterized by very rapid clinical process.Among more than a half of patients (about 59%) there is fever, weight loss or intensified, night hyperhidrosis [12,13].RS most commonly occurs in lymph nodes and bone marrow.Localization in lymphatic system occurs in about 41% of cases [14]. The disease described is a case of rare localization of transformation into RS in the course of CLL disease.Usually among CLL patients, the thyroid is secondarily infiltrated by leukemia cells [15].Lymphomas constitute only about 2% of all thyroid tumors, while those with the primary localization in this organ constitute only about 2.5% [16,17].Normal thyroid gland does not have lymphoid tissue.Its presence signifies ongoing pathological process, usually autoimmune inflammation.The literature presents very narrow number of patients with SLL/CLL and Hashimoto's inflammation or other tumors.We have not found a case of patient with Richter's transformation, either.Hashimoto's inflammation is often associated with occurrence of MALT type lymphoma (mucosa-associated lymphoid tissue; about 23% of lymphomas appearing on the thyroid) and DLBCL (most commonly occurring primary lymphoma of the thyroid, about 50% of cases) [18].Bocian presented patients among whom diagnosis of lymphoma had been preceded by autoimmune inflammation with the presence of high concentration of antibodies against peroxidase and thyroglobulin [21,22]. In the patient described in this case report, no preceding or accompanying inflammatory process was confirmed.Decrease in the concentration of FT3 and FT4 could be a result of so-called euthyroid sick syndrome.This phenomenon observed during numerous illnesses, including those related to tumours, however, is not accompanied by dysfunction of the organ itself [23]. In the available literature, only one case was found of RS localization (in the form of diffuse large B cell lymphoma) in liver, described by Tadmor et al.The case concerned a 72-year-old woman, initially treated with chlorambucil and prednisone.Analogously, as in the current case report, the authors observed a higher concentration of transaminases and a significant increase in LDH activity.There was also a very good reaction to immunochemistry therapy, which included vincristine, cyclophosphamide, doxorubicin, prednisolone and rituximab (R-CHOP) [24].Maryniak et al. presented a case where transformation into Hodgkin lymphoma in the course of CLL was confirmed only after the results of autopsy examination of liver, spleen, adrenal glands and bone marrow [25].Reddy et al., on the other hand, described HvRS in the liver of 59-year-old woman with a 10-year history of CLL, after many lines of cytostatic treatment (bendamustine, rituximab in monotherapy and with fludarabine).The first, quite unusual, clinical symptom of transformation was a significant increase of bilirubin and alkaline phosphatase concentration [26]. Of specific interest is a case presented by Salihoglu et al. which concerned a 52-year-old woman, where after observation and several more days of chemotherapy (chlorambucil, FC, R-COP), further progression of the disease was confirmed.Because of the detected deletion 17p, the patient was classified for allo-SCT, preceded by 6 CFAR chemotherapy courses (cyclophosphamide, fludarabine, alemtuzumab and rituximab).Clinical and immunophenotypic evaluation of peripheral blood and bone marrow before transplantation indicated complete remission.Repeated FISH test on the day 30 after allo-SCT did not indicate 17p deletion in bone marrow cells.However, 3 months after transplantation, sudden deterioration occurred in the patient's general health condition: diarrhea, skin lesions, ascites and significant increase of the liver size with the presence of numerous interstitial, focal lesions were all observed. On the basis of histopathological examination of sample taken during biopsy, large B cell lymphoma was diagnosed, which suggested transformation into RS.The patient died several weeks after confirming RS.According to the authors, the significant factor in transformation into RS, despite accompanying GVHD, was an unfavorable genetic profile of the patient [27]. Attention is being focused on a number of chemotherapy lines.Much controversy has arisen from an opinion about the possible influence of previous treatment for RS development, in particular, specific cytostatic drugs.CLL/SLL ill patients, during their illness usually receive many different types of chemotherapies, therefore the precise definition of a drug with specific significance in this matter is difficult.Modern drugs, including monoclonal antibodies (rituximab, alemtuzumab), may also probably be a significant factor in this process [28,29]. However, in the presented case report, as well as in the case described by Salihoglu et al., the patients are the proof that RS may occur at any moment in the course of the disease, even only after one type of chemotherapy.At the same time, this and other cases indicating the possibility of occurrence of a diffuse large B cell lymphoma among patients with CLL after allo-SCT, always require differentiation with possible post-transplantation lymphoproliferative disorder (PTLPD) [30,31]. ",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "Richter's syndrome is a rare complication of chronic lymphocytic leukemia, usually with an unfavourable prognosis.The liver and the thyroid are particularly rare locations of RS.On the basis of this case report and studies presented by other authors, it is not possible to clearly determine the influence of the site of transformation on the further course of disease, nor the factors influencing its development.However, such cases allow the conclusion that RS may occur even in the most unexpected and less probable place. ",
"section_name": "CONCLUSIONS",
"section_num": null
}
] |
[
{
"section_content": "",
"section_name": "Ministry of Science and Highe Education",
"section_num": null
},
{
"section_content": "",
"section_name": "Ministry of Science and Higher Education",
"section_num": null
},
{
"section_content": "",
"section_name": "Republic of Poland",
"section_num": null
},
{
"section_content": "",
"section_name": "Ministry of S and Higher E",
"section_num": null
},
{
"section_content": "",
"section_name": "Generation of the DOI (Digital Object",
"section_num": null
}
] |
10.3389/fonc.2020.540332
|
ST3Gal IV Mediates the Growth and Proliferation of Cervical Cancer Cells In Vitro and In Vivo Via the Notch/p21/CDKs Pathway
|
<jats:p>ST3Gal IV is one of the principal sialyltransferases responsible for the biosynthesis of α2, 3-sialic acid to the termini <jats:italic>N</jats:italic>-glycans or <jats:italic>O-</jats:italic>glycans of glycoproteins and glycolipids. It has been reported that ST3Gal IV expression is associated with gastric carcinoma, pancreatic adenocarcinoma and breast cancer. While the expression and functions of ST3Gal IV in cervical cancer are still poorly understood. In this study, we found that ST3Gal IV was downregulated in human cervical cancer tissues compared to normal cervix tissues, and ST3Gal IV expression was negatively associated with the pathological grade of cervical cancer. ST3Gal IV upregulation inhibited the growth and proliferation of cervical cancer HeLa and SiHa cells <jats:italic>in vitro</jats:italic> and <jats:italic>in vivo</jats:italic>. Furthermore, ST3Gal IV overexpression enhanced the expression of several Notch pathway components such as Jagged1, Notch1, Hes1 and Hey1, while cell cycle protein expression like Cyclin D1, Cyclin E1, CDK2 and CDK4 were decreased. These results indicate that expression of ST3Gal IV is reduced in cervical cancer and plays a negative role in cell proliferation <jats:italic>via</jats:italic> Notch/p21/CDKs signaling pathway. Thus, sialyltransferase ST3Gal IV might be a target for the diagnosis and therapy of cervical cancer.</jats:p>
|
[
{
"section_content": "Cervical cancer is one of the leading causes of cancer death among females, and its death rate ranks second among cancers in less developed countries (1).It has been demonstrated that cervical cancer is caused by human papillomavirus (HPV) infection (2).Significant advances have been made in cervical cancer screening and treatment.The development of HPV and its widespread adoption may contribute to reduced incidence of the disease.However, the incidence and mortality rates are still high due to the difficulties in achieving widespread compliance with HPV vaccination (3).In addition, HPV vaccines cannot protect against all the types of HPV that cause cervical cancer (1). Therefore, we focus more on the mechanism of cervical cancer occurrence and development, and try to find more ways to overcome cervical cancer. Sialylation is one of the most common glycosylation processes which adds sialic acids to the terminal of glycoconjugates.Current research suggests that sialylation is involved in various cellular functions such as cell adhesion and signal transduction.Sialyltransferases (STs) include 20 members and are divided into four families: ST3Gal I-VI, ST6Gal I-II, ST6GalNAc I-VI, and ST8Sia I-VI (4).These sialyltransferases are responsible for the transfer of sialic acid and participate in the post-translational modifications of proteins.There is increasing evidence that abnormal expression levels of sialyltransferases are related to tumor proliferation, invasion, metastasis and drug resistance (5)(6)(7). ST3Gal IV is one of the major enzymes responsible for the formation of a2, 3-sialylated N-glycan or O-glycan (8,9).It has been reported that the expression of ST3Gal IV is associated with various tumors such as gastric cancer, head and neck squamous cell carcinoma (HNSCC) (10) and renal cell carcinoma.Peŕez-Garay et al. demonstrated that ST3Gal IV was highly expressed in pancreatic adenocarcinoma tissues and enhanced the migration and metastasis of pancreatic adenocarcinoma cells (11).Saito et al. found that the mRNA level of ST3Gal IV in primary renal cell carcinoma tissues was decreased compared with non-tumor kidney tissues, and that the down-regulated ST3Gal IV was considered to be one of the factors related to the malignant progression of renal cell carcinoma (12).However, whether ST3Gal IV is involved in the proliferation of cervical cancer cells and the mechanisms behind these observations remain poorly understood. In this study, combined with cervical cancer tissue chips and clinical baseline data analysis, we found that the expression level of ST3Gal IV in cervical cancer tissues was lower than that in adjacent tissues, and was negatively correlated with the malignancy of the tumor.The overexpression of ST3Gal IV in cervical cancer cells inhibited the proliferation, migration and invasion capability of SiHa and HeLa cells.ST3Gal IV overexpression inhibited the proliferation and tumorigenesis of HeLa cells in nude mice.In addition, the expression levels of Jagged1, Notch1, Hes1 and Hey1 in Notch signaling pathway were increased upon ST3Gal IV upregulation, while p21, CyclinD1, CyclinE1, CDK2 and CDK4 protein levels were decreased.Moreover, Notch inhibitor DAPT restored the expression levels of above-mentioned proteins in cells with overexpression of ST3Gal IV.In conclusion, ST3Gal IV expression inhibits the growth and proliferation of cervical cancer cells in vitro and in vivo through the Notch1/p21/CDKs signaling pathway. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "Cervical carcinoma cell lines SiHa and HeLa were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Novato, CA, United States).The medium contains 10% fetal bovine serum (Gibco, Novato, CA, United States), 100 U/ml penicillin, and 100 U/ml streptomycin (Beyotime, China).Then the cell lines were cultured in cell incubator at 37°C with 5% CO 2 . ",
"section_name": "Cell Culture",
"section_num": null
},
{
"section_content": "Tumor tissues from xenograft model and commercial tissue arrays (Superbiotek, Shanghai, China) were dewaxed in dimethylbenzene and hydrated in gradient alcohol.After antigen retrieval and blocking, primary antibody (1:80; Proteintech, 13546-1-AP) against ST3Gal IV was incubated with the tissue samples overnight at 4°C.Then the biotinylated secondary antibody was used to combine with the primary antibodies and incubated for 30 min at 37°C.Diaminobenzidine (DAB) (ZSGB-BIO, Beijing, China) was used as a developer, and hematoxylin was used for counterstaining.Two observers reviewed the immunohistochemical staining results independently and assessed the staining intensity and extent by German semi-quantitative scoring system (13).The cut-off value for differentiating between final positive and negative immunostaining was set at 4 by using the receiver operating characteristic (ROC) curve analysis (14,15).Score of 0 to 3 points was considered negative for protein expression and score of 4 points or greater was considered positive which showed high protein expression. ",
"section_name": "Immunohistochemistry",
"section_num": null
},
{
"section_content": "HeLa and SiHa cells were treated with a mixture of recombinant pcDNA3.1/ST3GalIV vector or a control vector and Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) depending on the manufacturer's instructions to increasing the expression of ST3Gal IV or as a control group.After 48 h, 600 µg/ml, 800 µg/ml of G418 (Sigma-Aldrich, Darmstadt, Germany) were used to select stably transfected HeLa and SiHa cells respectively.The expression of ST3Gal IV was confirmed by RT-PCR, Western blot and Lectin blot. ",
"section_name": "Cell Transfection",
"section_num": null
},
{
"section_content": "Total RNA was extracted from normal, control and transfected HeLa, SiHa cells by TRIzol reagent (Life Technologies, Carlsbad, CA, USA) and employed to synthesize cDNA using a PrimeScript RT Reagent Kit (Takara, Dalian, China).Subsequently, cDNA mixed with qPCR SuperMix (Takara, Otsu, Japan) and GAPDH or ST3Gal IV primer (GenePharma) then reacted at 94°C for 3 min, 94°C for 5 s and 60°C for 34 s with 40 cycles.The expression levels were analyzed using the 2 -DDCT method and the GAPDH was used as an internal control. ",
"section_name": "Real-Time PCR",
"section_num": null
},
{
"section_content": "Proteins were extracted from cells and determined by BCA kit (Beyotime).Equal amounts of protein were separated by 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred onto PVDF membranes (Pall Corporation, New York, NY, USA).Then we blocked the membranes with 5% skim milk in TBST at room temperature for 2 h.The membranes were incubated with primary antibodies of ST3Gal IV (1:1000, proteintech, 13546-1-AP), Jagged1 (1:750, Elabscience, ENK5401), Notch (1:750, proteintech, 20687-1-AP), Hes1 (1:1000, Elabscience, EAP2709), Hey1 (1:1250, proteintech, 19929-1-AP), p21 (1:1000, Bioworld, BS6501), p53 (1:1000, Bioworld, BS3156), Cyclin D1 (1:1000, Affinity biosciences lnc, DF6386), Cyclin E1 (1:1000, Bioworld, BZ00342), CDK2(1:1000, Bioworld, BS1050), CDK4 (1:1000, Bioworld, BS6462), GAPDH (1:4000, proteintech, 10494-1-AP) overnight at 4°C.Subsequently, these membranes were incubated with secondary antibody (1:10,000, ZSGB-BIO, ZB-2301) for 2 h at room temperature.The protein bands on the membranes were visualized by ECL kit (Advansta, Menlo Park, CA, USA) and conducted with the Image Lab software (Bio-Rad, Hercules, CA, USA). ",
"section_name": "Western Blot Assay",
"section_num": null
},
{
"section_content": "Similar to the Western blot assay, about 30 mg of protein was loaded onto two 10% SDS-polyacrylamide gels while one was subjected to Coomassie Brilliant Blue (CBB) staining and the other was transferred onto PVDF membranes.The membrane was blocked in 5% skim milk at room temperature for 2 h and incubated with Maackia Amurensis Lectin II (MAL II, 1:1000, Vector Laboratories, B-1265) or biotin-labeled Lectin Sambucus nigra agglutinin (SNA, 1:2000, Vector) for 2 h at 37°C.Then the membrane was incubated with horseradish peroxidase streptavidin (1:1000, ZSGB-BIO) for an hour at room temperature.Image Lab software (Bio-Rad) was used for detection and Gel-Pro software worked as analyzer. ",
"section_name": "Lectin Blot Analysis",
"section_num": null
},
{
"section_content": "Cell membrane proteins were extracted using a CelLytic MEM Protein Extraction kit (Sigma) according to the manufacturer's instruction.The proteins (200 mg per group) were mixed with 50 mM NH 4 HCO 3 and 50 mM dl-dithiothreitol (DTT) and reduced at 56°C for 30 min.The alkylation was carried out by adding 50 mM iodoacetamide followed by incubation in dark at room temperature for 1 h.The mixtures were then treated with trypsin (2 mg) at 37°C for 16 h, and inactivation at 95°C for 5 min.The sample was finally incubated with 3 mU peptide N-glycanase F (PNGase F) (Takara) at 37°C overnight and vacuum dried.The released N-glycans were purified using an Oasis MCX cartridge (30 mg/ml; Waters) and lyophilized. Permethylation of the freeze-dried N-glycans was performed according to the method described previously (16) with some minor changes.Dimethyl sulfoxide (DMSO) containing 1% v/v distilled water (50 ml) was added to the dried glycan sample under alkaline conditions with powdered sodium hydroxide.The mixtures were treated with Methyl iodide (50 ml) at room temperature for 30 min with vigorous shaking.Distilled water (1 ml) was slowly added to the reaction mixture, and then the mixture was applied to a Sep-Pak Vac 18 cartridge (50 mg/ml; Waters).Finally, the permethylated glycans were eluted with 80% v/v acetonitrile in distilled water and evaporated to dryness. ",
"section_name": "Purification of N-Glycans from Cell Membrane Proteins",
"section_num": null
},
{
"section_content": "MS Spectra of the permethylated glycans were acquired using a matrix-assisted laser desorption/ionization-time of flight/time of flight (MALDI-TOF/TOF) MS (New ultrafleXtreme, Bruker Daltonik).Ions were generated by signal averaging over 4,000 laser shots from a Smartbeam-II Nd : YAG laser operating at 355 nm and a repetition rate of 2 kHz.All spectra were obtained in the reflectron mode with delayed extraction of 200 ns.For sample preparation, 0.5 ml of 2, 5-dihydroxybenzoic acid (DHB, 10 mg/ml) in 30% ethanol was spotted onto a target plate (MTP 384 target plate ground steel, Bruker Daltonik).After dried, an aliquot (0.5 ml) of the glycan solution was spotted onto the DHB crystal and dried.Mass spectra were obtained from Na + adductions. ",
"section_name": "Mass Spectrometry (MS)",
"section_num": null
},
{
"section_content": "Cells were plated in 96-well plates at a density of 4 × 10 3 cells per well in triplicate and 10 ml of CCK-8 reagent was added into each well and incubated at 37°C with 5% CO 2 for an hour.Then the microplate reader (Thermo Fisher Scientific) was used to measure the absorbance at 450 nm.Moreover, 1 × 10 3 cells per well were seeded in 6-well plates and incubated for two weeks.Subsequently, cells were fixed with methanol and stained with 0.1% crystal violet.The cell clusters comprised more than fifty cells. ",
"section_name": "Cell Survival Assays by Cell Counting Kit-8 and Cell Colony Formation Assay",
"section_num": null
},
{
"section_content": "Cells were gathered as single cell suspension and fixed with 70% alcohol at 4°C overnight.Then centrifuged for 5 min at 1,000 rpm and washed two times by cold PBS liquid to get rid of alcohol.Cells were re-suspended in PBS containing 50 mg/ml RNase and 50 mg/ml PI (propidium iodide) and incubated for 30 min at 4°C in the dark.RNase is used to decompose RNA in cells and PI for the staining of DNA.Subsequently, flow cytometer (FACSCalibur, BD, USA) with CELLquest pro software analyzed the cell cycle distribution by detecting the fluorescence intensity of PI. ",
"section_name": "Flow Cytometry",
"section_num": null
},
{
"section_content": "EdU (5-ethynyl-2′-deoxyuridine) as a nucleoside analog of thymidine could involve in the DNA synthesis then measure the cell proliferation ability (17) and reflect cell cycle distribution.Cells were plated on slips at appropriate density for 12 h and treated with EdU kit (Invitrogen, USA) according to the manual.The fluorescence was observed by confocal laserscanning microscope (BD, Biosciences). ",
"section_name": "EdU Assay",
"section_num": null
},
{
"section_content": "Nude mice aged 4 to 6 weeks were obtained from the Animal Experiment Center of Dalian Medical University and divided into a HeLa group and two HeLa/ST3Gal IV groups randomly.2 × 10 7 cells were injected into the right dorsal flank of each mouse and the tumor diameters were measured by vernier caliper every 5 days a week later.After a month, all mice were killed and the weights of tumors were determined, and the volumes were calculated by the formula: 1/2 (length × width 2 ) (18). ",
"section_name": "Xenograft Model",
"section_num": null
},
{
"section_content": "Data were presented as mean ± SD and analyzed with SPSS 20.0 (SPSS Inc., USA) and each assay was performed at least three times independently.The differences between two groups were determined by Student's t-test and among three or four groups were analyzed by one-way ANOVA.P < 0.05 was considered to be statistically significant. ",
"section_name": "Statistical Analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "To analyze the expression levels of ST3Gal IV in the development of cervical cancer, 75 cases of cervical cancer tissue microarray were evaluated by immunohistochemistry (IHC).As shown in Figure 1A, the expression of ST3Gal IV decreased with the increase of the malignancy of cervical cancer.Box plots of IHC scores for ST3Gal IV expression also showed that ST3Gal IV expression was lower in cervical cancer tissue compared to normal cervical tissue (Figure 1B). According to the IHC score, 70 cervical cancer cases were subdivided into a \"low ST3Gal IV expression\" group containing 36 samples (score of 0 to 3) and a \"high ST3Gal IV expression\" group containing 34 samples.Five cases of normal tissues did not participate in statistics.The association between ST3Gal IV expression and patient ages, pathological types, pathological grading, primary tumor stages and lymph node metastasis was assessed by Pearson's chi-squared test (Figure 1C).These results suggest that ST3Gal IV expression was negatively correlated with pathological grading of cervical cancer tissues (p = 0.005). ",
"section_name": "ST3Gal IV Expression Is Negatively Related to the Malignant Degree of Cervical Cancer Tissues",
"section_num": null
},
{
"section_content": "To further explore whether ST3Gal IV is associated with the development of cervical cancer, the expression of ST3Gal IV was up-regulated in two typical cervical cancer cell lines, HeLa and SiHa cells.Real-time PCR results showed that the mRNA levels of ST3Gal IV were increased significantly compared to the control and mock cells (Figures 2A,B).Meanwhile, ST3Gal IV protein expression was increased (Figures 2C,D).Furthermore, Lectin blot analysis (Figures 2E-H) showed that ST3Gal IV overexpression enhanced the expression of a2, 3-linked sialic acid and decreased the expression levels of a2, 6-linked sialic acid in HeLa and SiHa cells.These results suggest that ST3Gal IV can be stably overexpressed in HeLa and SiHa cells by transfecting the overexpression vector. ",
"section_name": "Establishment of Stable ST3Gal IV-Upregulated Cervical Cancer Cell Lines",
"section_num": null
},
{
"section_content": "To determine whether the profiles of N-glycan are altered in cervical cancer cells after overexpression of ST3Gal IV, we compared Nlinked glycans on cell membranes produced by HeLa, HeLa/ST3Gal IV, SiHa, and SiHa/ST3Gal IV cells.The MS spectrum and the monosaccharide composition for each signal were summarized in Figure 3 and Table 1, respectively.In total, 45 kinds of glycoforms were detected and relatively quantified in these four cell lines.Nglycan compositions with the most intense signal detected in HeLa cells was peak 15, followed by 8, 24, 4, 29, 19, 16 (in the order of decreasing signal strength), while in HeLa/ST3Gal IV cells was peak 29, followed by 24, 15, 8, 16, 7, 4.Among these N-linked glycans, peak 29 (m/z = 2966.18)[(Hex) 2 (HexNAc) 2 (Fuc) 1 (NeuAc) 2 +(Man) ",
"section_name": "MALDI-TOF MS Analysis for N-glycan Profiling of Cervical Cancer Cell Lines",
"section_num": null
},
{
"section_content": "To investigate the function of ST3Gal IV in the malignant phenotype of cervical cancer cells, CCK8, colony formation and EdU assays were performed to detect the cell proliferation 4E,F).Furthermore, EdU assay showed that ST3Gal IV overexpression inhibited the DNA synthesis in HeLa and SiHa cells (Figures 4G-I) compared to the control groups.Together, our findings suggest ST3Gal IV overexpression has a negative effect on cell proliferation and colony formation ability of HeLa and SiHa cells in vitro. ",
"section_name": "ST3Gal IV Overexpression Inhibits the Proliferation and Colony Formation Ability of HeLa and SiHa Cells In Vitro",
"section_num": null
},
{
"section_content": "To investigate the effects of ST3Gal IV upregulation on the proliferation of HeLa and SiHa cells, flow cytometry assay was performed to detect the cell cycle distribution.By calculating the number of cells in each cell cycle, we found that overexpression of ST3Gal IV in cervical cancer cells resulted in increased S phase cell populations and decreased G2/M cell populations, which was contrast with that in control groups (Figures 5A,B).This suggests that ST3Gal IV may mediate the proliferation of cervical cancer cells by affecting DNA synthesis.A large number of studies have revealed that Notch1 signaling pathway exerts great influence in tumor development.To elucidate whether this pathway was involved in ST3Gal IV-mediated cell proliferation, we analyzed the expression levels of related proteins in Notch1 signaling pathway.The results showed that the expression of Jagged1, Notch1, Hes1, Hey1 and p21 was significantly increased meanwhile CyclinD1, CyclinE1, CDK2, CDK4 expression levels were decreased observably in ST3Gal IV overexpressing cells compared to control cells (Figures 5C,D).However, there was no apparent change in the expression levels of p53 in ST3Gal IV overexpressing cells.These results indicate ST3Gal IV overexpression can induce cell cycle arrest in S phase and may affect cell proliferation through the Notch1/p21/CDKs signaling pathway. ",
"section_name": "Upregulation of ST3Gal IV Blocks Cell Cycle in S Phase and Influences Notch1/p21/CDKs Signaling Pathway in Cervical Cancer Cells",
"section_num": null
},
{
"section_content": "To further clarify that ST3Gal IV can mediate the growth and proliferation of cervical cancer cells through the Notch pathway, ST3Gal IV overexpressing cells and mock cells were treated with Notch1 inhibitor DAPT.As shown in Figures 6A,B, in mock/ DAPT group, DAPT treatment significantly reduced the expression levels of Jagged1, Notch1, Hes1, Hey1, p21 and increased CyclinD1, CyclinE1, CDK2, CDK4 expression.However, there was no significant difference in p53 expression between the mock/DAPT group and ST3Gal IV/DAPT group.In addition, we found that the expression levels of the above-mentioned proteins in the ST3Gal IV/ DAPT group were consistent with those in mock cells.This result suggests DAPT treatment can restore the expression levels of proliferation related proteins in ST3Gal IV upregulating cervical cancer cells.Taken together, these results confirm that ST3Gal IV overexpression inhibits the proliferation of cervical cancer cells via the Notch1/p21/CDKs signaling pathway. ",
"section_name": "Notch Inhibitor DAPT Restores the Expression Levels of Proliferation-Related Proteins in ST3Gal IV Overexpression Cells",
"section_num": null
},
{
"section_content": "To determine the effect of ST3Gal IV overexpression on the tumorigenic ability of cervical cancer cells in vivo subdermal injection of nude mice with HeLa cells overexpressing ST3Gal IV or carrying a control vector.The xenograft tumor size was dramatically decreased in the ST3Gal IV upregulation groups a) The glycans were calculated as permethylated glycan and [M + Na] + .b) Monosaccharide compositions were determined by database searching using GlycoMod (http://www.expasy.ch/tools/glycomod/). Monosaccharides are indicated as follows: Hex, hexose; HexNAc, N-acetyl hexosamine; Fuc, fucose; NeuAc, N-acetyl neuraminic acid; Man, mannose. compared to mock group (Figures 7A,B).The average tumor weight and approximate tumor volume of the HeLa/ST3Gal IV tumors were significantly decreased compared with those in mock group (Figures 7C,D).Further, ST3Gal IV overexpression increased the Jagged1, Notch1, Hes1, Hey1 and p21 expression, meanwhile, decreased the expression levels of CyclinD1, CyclinE1, CDK2 and CDK4 in xenograft tumor tissues (Figures 7E-G).Therefore, these findings demonstrate that ST3Gal IV might be involved in the tumorigenesis and progression of cervical cancer via Notch1/p21/CDKs pathway in vivo. ",
"section_name": "ST3Gal IV Overexpression Inhibits the Tumorigenicity of HeLa Cells in Nude Mice Through Notch1/p21/CDKs Pathway",
"section_num": null
},
{
"section_content": "Abnormal sialylation has been reported to impact tumor cell growth, differentiation, adhesion and invasion (19)(20)(21).In this study, we detected the expression level of ST3Gal IV in cervical cancer tissue microarray (n = 75), finding low ST3Gal IV expression in the cervical cancer tissues compared to normal cervix tissues.The results were consistent with those reported by Wang et al. that ST3Gal IV mRNA expression levels were downregulated in cervix squamous cell carcinoma (22).However, there is little research about the role of ST3Gal IV in cervical cancer progression.Subsequently we analyzed the correlation between ST3Gal IV expression and the pathological grade of cancer tissues, and statistical results showed that low expression of ST3Gal IV was dramatically related to the high malignant degree of cervical cancer tissues.Our findings indicated ST3Gal IV may act as a tumor suppressor in cervical cancer.Previous studies suggested that a2, 3 sialyltransferase and a2, 6 sialyltransferase compete with common substrate (8,23).Consistent with these findings, the Lectin blot analysis showed that the overexpression of ST3Gal IV can decrease expression of a2, 3-linked sialic acid in cervical cancer cells.However, how the decrease of a2, 6 sialylation affects the growth and proliferation of cervical cancer cells is what we need to explore in the next research. The results of Mass Spectrometry suggested that high mannose is the main N-glycan components in any kind of cell lines we used, which is consistent with the N-glycosylation characteristics of cell membrane proteins.Although HeLa and SiHa are both cervical cancer cell lines, the different origins lead to slight differences in glycan pattern between them, which is mainly reflected in the different glycan pattern between peak 16 and 45.After the overexpression of ST3Gal IV, the glycan pattern of these two cell lines have changed observably, especially the signal intensity of the four polysaccharides of peak 29,36,40,45 increased significantly.The results indicated the transfection of ST3Gal IV is successful since the four polysaccharides contained sialic acid residues.Although our experimental results could not expression in total glycan increased significantly, and the results of repeated experiments were consistent with this.However, this phenomenon was not found in SiHa/ST3Gal IV cell line and the specific reason is not clear and needs further study.It has been reported that ST3Gal IV is up-regulated and plays a pro-cancer role in numerous cancers including pancreatic adenocarcinoma, gastric cancer and leukemia (11,(24)(25)(26).However, our study found that upregulation of ST3Gal IV inhibited cervical cancer cell proliferation in vitro and mediated cell cycle arrest in S phase.Furthermore, the in vivo xenograft experiment also suggested that HeLa cells with high ST3Gal IV expression showed lower tumorigenicity rates and growth than that in mock group.We speculate that this may be caused by epigenetic changes, such as DNA hypermethylation.As Kawamura YI reported that the hypermethylation of ST3Gal VI gene induces the aberrant glycosylation and expression of carbohydrate antigens by silencing of the activity of glycosyltransferases in gastrointestinal cancer (27).Therefore, whether ST3Gal IV has undergone the epigenetic changes in cervical cancer cells still needs to be explored in our future work.In addition, the difference between subcutaneous microenvironment and cervical environment in nude mice is one of the limitations of in vivo experiment.Another limitation is the presence of N-acetylneuraminic acid (Neu5Ac) and Nglycolylneuraminic acid (Neu5Gc) in nude mice, while humans only contain Neu5Ac because of a mutation in CMP-sialic acid hydroxylase (28).These make some errors in our experiment and we look forward to building a more rigorous xenograft model in our next experiment. Recent findings have revealed a variety of molecular events involved in cervical carcinogenesis, such as Wnt signaling, C-Met/HGF signaling pathway, PI3K/AKT/mTOR signaling pathway, etc. (29)(30)(31).Notch signaling was involved in cell proliferation, differentiation and apoptosis as a mechanism for evolutionarily conserved intercellular interaction.The overexpression of Notch1 signaling proteins in cervical cancer suggested that Notch1 may promote tumor progression (32)(33)(34).However, the effect of Notch signaling on cervical cancer is not entirely clear.Other studies demonstrated that the activation of Notch signaling displayed the suppression on cancer cells proliferation (35,36).In our study, ST3Gal IV overexpression induced the up-regulation of Notch1, the cognate ligand, Jagged1 and the Notch downstream targets Hes1 and Hey1.The tumor suppressor p21, one of the Notch1 target genes (37), was also increased in cervical cancer cells overexpressed by ST3Gal IV.Furthermore, consistent with the fact that p21 is a cyclindependent kinase inhibitor, we found that levels of cyclin- Notch protein undergoes post-translational modification to accomplish its activity and functions, and glycosylation is a significant factor regulating Notch signaling pathway (38)(39)(40).Several extracellular epidermal growth factor-like (EGF) domains of Notch are modified with O-fucose and O-glucose glycans as well as N-glycans that alter receptor sensitivity to ligand stimulation (38,41).These studies indicated that Nglycosylation and O-glycosylation played essential roles in Notch1 signaling pathway.Our result revealed that ST3Gal IV overexpression caused a significant increase in the expression of levels of Jagged1, Notch1 and the downstream molecules, and the upregulation of p21 inhibited the expression of CDKs, suggesting that ST3Gal IV might participate in the modulation of Notch receptors or ligands in cervical cancer cells.There are many crosstalks between Notch signals and other signaling pathways (such as TGF-b/Smad, Wnt, NF-kB, PI3K/Akt, etc.) (42)(43)(44)(45)(46). Therefore, further studies are needed to elucidate the precise molecular mechanisms by which ST3Gal IV modulates the Notch1/p21/CDKs signaling pathway in cervical cancer.Taken together, our research indicated that ST3Gal IV was negatively associated with the pathological grade of cervical cancer tissues, and the overexpression of ST3Gal IV inhibited the growth and proliferation of cervical cancer cells in vivo and in vitro through Notch1/p21/CDKs signaling pathway.Therefore, ST3Gal IV might be a potential target for the prognosis determination and treatment of cervical cancer in the future. ",
"section_name": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "FUNDING This work is supported by Natural Science Foundation of Liaoning Province and Transverse Program of Dalian Medical University (2019-ZD-0640, 505526) and the Liaoning Provincial Program for Top Discipline of Basic Medical Sciences. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The datasets generated for this study are available on request to the corresponding authors. ",
"section_name": "DATA AVAILABILITY STATEMENT",
"section_num": null
},
{
"section_content": "All care and experimental procedures were performed in accordance with the guidelines approved by the Dalian Medical University Animal Ethics Committee (Animal protocol No. AEE18067).In addition, all animal experiments in the present study were consistent with the National Institutes of Health guide for the care and use of laboratory animals. SJW, XS, YSW, and XC designed the study.YSW, XC, and WD contributed equally to this study and performed all experiments.WD and YW completed the mass spectrometry analysis experiment.WD, YW, and GL analyzed the mass spectrometry analysis data.YJ, ZX, CX, and LZ participated in cell culture and cell transfection experiments.AW analyzed the association between ST3Gal IV expression and clinical data.XY, XS, and SDW participated in the tissues detection.SJW, YSW, and XC wrote and revised the manuscript.All authors contributed to the article and approved the submitted version. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2020.540332/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. ",
"section_name": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "All care and experimental procedures were performed in accordance with the guidelines approved by the Dalian Medical University Animal Ethics Committee (Animal protocol No. AEE18067).In addition, all animal experiments in the present study were consistent with the National Institutes of Health guide for the care and use of laboratory animals. ",
"section_name": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "SJW, XS, YSW, and XC designed the study.YSW, XC, and WD contributed equally to this study and performed all experiments.WD and YW completed the mass spectrometry analysis experiment.WD, YW, and GL analyzed the mass spectrometry analysis data.YJ, ZX, CX, and LZ participated in cell culture and cell transfection experiments.AW analyzed the association between ST3Gal IV expression and clinical data.XY, XS, and SDW participated in the tissues detection.SJW, YSW, and XC wrote and revised the manuscript.All authors contributed to the article and approved the submitted version. ",
"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/fonc.2020.540332/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. ",
"section_name": "SUPPLEMENTARY MATERIAL",
"section_num": null
}
] |
10.1038/s41598-020-70790-9
|
Mediating effect of soluble B-cell activation immune markers on the association between anthropometric and lifestyle factors and lymphoma development
|
<jats:title>Abstract</jats:title><jats:p>Sustained B-cell activation is an important mechanism contributing to B-cell lymphoma (BCL). We aimed to validate four previously reported B-cell activation markers predictive of BCL risk (sCD23, sCD27, sCD30, and CXCL13) and to examine their possible mediating effects on the association between anthropometric and lifestyle factors and major BCL subtypes. Pre-diagnostic serum levels were measured for 517 BCL cases and 525 controls in a nested case–control study. The odds ratios of BCL were 6.2 in the highest versus lowest quartile for sCD23, 2.6 for sCD30, 4.2 for sCD27, and 2.6 for CXCL13. Higher levels of all markers were associated with increased risk of chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL). Following mutual adjustment for the other immune markers, sCD23 remained associated with all subtypes and CXCL13 with FL and DLBCL. The associations of sCD23 with CLL and DLBCL and CXCL13 with DLBCL persisted among cases sampled > 9 years before diagnosis. sCD23 showed a good predictive ability (area under the curve = 0.80) for CLL, in particular among older, male participants. sCD23 and CXCL13 showed a mediating effect between body mass index (positive) and DLBCL risk, while CXCL13 contributed to the association between physical activity (inverse) and DLBCL. Our data suggest a role of B-cell activation in BCL development and a mediating role of the immune system for lifestyle factors.</jats:p>
|
[
{
"section_content": "B-cell lymphomas (BCL) are an etiologically, clinically, and histologically heterogeneous group of malignant diseases of B lymphocytes.Immunodeficiency and autoimmunity are strong B-cell lymphoma risk factors.Chronic B-cell activation is suspected to be an important mechanism contributing to the accumulation of genetic errors that can lead to lymphomagenesis 1 .Increased serum/plasma levels of molecules involved in B-cell activation, among which soluble (s)CD23, sCD27, sCD30, sCD44, and CXCL13, have been associated with the development of acquired immune deficiency syndrome (AIDS)-related BCL [2][3][4][5][6][7] .Recently, studies within general population cohorts incorporating serologic measurements of cytokines, chemokines, and other immune markers have provided important evidence supporting a role for subtle immunologic effects in lymphomagenesis even among non-immunocompromised individuals [8][9][10][11][12] .Elevated serum levels of sCD23, sCD27, sCD30, and CXCL13 have subsequently been shown to be associated with BCL development in immunocompetent individuals [8][9][10][11][12] . The risk of BCL has been associated with lifestyle, viral, and environmental factors 13,14 .A large study from the International Lymphoma Epidemiology Consortium (InterLymph) showed several risk/protective factors for Non-Hodgkin lymphoma subtypes 13 .These included a family history of hematologic malignancy, history of autoimmune disease, hepatitis virus C (HCV) infection, body mass index (BMI), height, smoking, and occupation, which were all associated with increased risk of NHL and/or one of its subtypes (CLL, FL, or DLBCL), while alcohol intake (≥ 1 drink per month), better socio-economic position (SEP), history of atopic disease, and recreational sun exposure were linked with reduced risks.Given the central role of the immune system in lymphomagenesis, most risk factors may influence BCL risk through modulation of the immune function 15 .Indeed, diet and obesity have an important influence on the immune system as immune functions are sensitive to both under-and over-nutrition 16 .Obesity promotes increased production of cytokines and leptin 16 , and the latter, in turn, has been shown to enhance B-cell survival 17 . Infection with hepatitis virus B (HBV) and HCV correlate with high sCD30 [18][19][20] and sCD23 21,22 serum levels.In a study including HCV-infected patients, with and without BCL, a signature involving sCD27, sIL-2Rα, gamma globulins, and complement factor 4 was associated with the presence of overt BCL in HCV-infected patients 23 . Moreover, exposure to environmental factors suspected to be acting as lymphomagens (i.e.trichloroethylene and 2,3,7,8-tetrachlorodibenzo-p-dioxin) altered sCD27 and sCD30 plasma levels 24,25 .Studies have shown that alcohol consumption may be associated with a decreased risk of BCL 13 .Alcohol is associated with dysregulation of cytokines and chemokines, which may mediate alcohol-induced tumor promotion.Alcohol may also affect transcription factors and signaling pathways that regulate the expression/function of cytokines and chemokines 26 .Alcohol abuse impairs both the number and function of B cells.Chronic alcohol consumption reduces B-cell numbers, decreases antigen-specific antibody responses, increases the production of auto-antibodies, and interferes with B-cell development and maturation.Moreover, alcohol's impact on T and B cells increases the risk of infections (e.g., pneumonia, HIV infection, hepatitis C virus infection, and tuberculosis) 27 .However, to date, the direct mediating effect of immune markers on the association between these factors and BCL risk has not been investigated in a prospective setting. In our previous study among participants of two prospective cohorts (32 BCL cases overlap with cases included in the present study), which also included a meta-analysis of published data on sCD27 and sCD30, we reported a highly consistent association between these markers and increased risk of BCL subtypes 12 .Prediagnostic sCD23 was a strong predictive marker (area under the curve (AUC) = 0.88) for diagnosis of CLL ( 179MAP5 UMR 8145, Université de Paris, 75006 Paris, France. 11CESP, Fac. de Médecine -Univ.Paris-Sud, Fac de Médecine -UVSQ, INSERM, Université Paris Saclay, 94805 Villejuif, France. 12Gustave Roussy, 94805 Villejuif, France. 13Department of Hematology and Oncology, Hospital of Versailles, Le Chesnay, France. 14Cancer Risk Factors and Life-Style Epidemiology Unit, Institute for Cancer Research, Prevention and Clinical Network -ISPRO, Florence, Italy. 15Epidemiology and Prevention Unit, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milan, Italy. 16Department of Clinical and Biological Sciences, University of Turin, Turin, Italy. 17Unit of Epidemiology, Regional Health Service ASL, Turin, Italy. 18Department of Epidemiology, Murcia Regional Health Council, IMIB-Arrixaca, Murcia, Spain. 19CIBER of Epidemiology and Public Health (CIBERESP), Madrid, Spain. 20Andalusian School of Public Health (EASP), Granada, Spain. 21Instituto de Investigación Biosanitaria de Granada (Ibs.GRANADA), Universidad de Granada, Granada, Spain. 22Unit of Nutrition, Environment and Cancer, Cancer Epidemiology Research Program and Translational Research Laboratory, Catalan Institute of Oncology (ICO), Biomedical Research Institute (IDIBELL), Barcelona, Spain. 23Hellenic Health Foundation, Athens, Greece. 24Pulmonary Medicine Department, School of Medicine, National and Kapodistrian University of Athens, \"ATTIKON\" University Hospital, Haidari, Greece. 25Department of Clinical Sciences and Community Health Università Degli Studi di Milano, 20133 Milan, Italy. 26Division of Cancer Epidemiology, German Cancer Research Center (DKFZ), Heidelberg, Germany. 27Research Group Genomic Epidemiology, German Cancer Research Center (DKFZ), Heidelberg, Germany. 28Department of Epidemiology and Biostatistics, School of Public Health, Imperial College London, London, UK. 29 Department of Nutrition, Bjørknes University College, Oslo, Norway. 30Department of Endocrinology, Morbid Obesity and Preventive Medicine, Oslo University Hospital, Oslo, Norway. 31Department of Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany. 32Department of Molecular Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany. 33Institute of Nutritional Sciences, University of Potsdam, Nuthetal, Germany. 34Cancer Epidemiology Unit, Nuffield Department of Population Health, University of Oxford, Oxford, UK. 35 Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht, The Netherlands. 36Department of Epidemiology and Biostatistics, MRC-PHE Centre for Environment and Health, Imperial College London, London, UK. * email: [email protected] cases overlap with cases included in the present study) 28 .So far, previous studies have limitations in terms of sample size, in particular for BCL subtypes, limiting their power to detect associations of moderate magnitude. In the present study, we aimed to extend our previous findings using a much larger population within the European Prospective Investigation into Cancer and Nutrition (EPIC) population to examine the relationship between pre-diagnostically level of the most promising previously reported B-cell activation markers (sCD23, sCD27, sCD30, and CXCL13) and subsequent development of BCL major subtypes, i.e. diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL).Moreover, we aimed at exploring the potential clinical utility of these markers for screening and their possible mediating effects on the association between anthropometric and lifestyle factors and BCL subtypes. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Table 1 shows the characteristics of the study population.Overall, case and control subjects did not differ with regard to risk factors and covariates.Median time between recruitment (i.e., blood collection) in the study and diagnosis of BCL was 9 years (range, 0.2-19).Blood levels of all B-cell activation markers were significantly higher in cases compared to controls (Table 1).Spearman correlations were calculated between the various Table 1.General characteristics of B-cell lymphoma cases and controls.a Mean (SD); Body mass index (BMI); diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL).b one CLL and 9 controls lacked paired samples ; Due to limited sample volume, sCD27 and CXCL13 were not measured in 5 and 11 samples, respectively.So sample size for those markers are as: Matched analyses: CD27 (511 pairs), CXCL13 (506 pairs).Unmatched analyses: CD27 (515 cases/522 controls), CXCL13 (511/520).c Median (min-max).Bold: statistically significant at p-value ≤ 0.05.1).We found the lowest correlations between these immune markers among controls, while sCD30 was highly correlated with sCD27 in CLL, and with all other markers in FL.Levels of the markers were positively correlated with age in both cases and controls except for CXCL13 in control subjects (Supplementary Fig. 1).Compared to male participants, female subjects had slightly higher levels of sCD30 and CXCL13 that met the threshold of statistical significance (Supplementary Table 2).There was no significant difference in levels of the markers in different countries except for CXCL13 (Supplementary Table 2). ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "logistic regression analyses for all BCL cases showed a significant association for all markers when analyzed as categorized variables (Table 2).The subtype-specific analyses rendered similar results except for a non-significantly increased risk of CLL with elevated levels of CXCL13 (Table 2).To account for the correlation between the markers, all markers were also modeled together.The combined multivariable models showed a significant association between sCD23 and all three BCL subtypes, and between CXCL13 and FL and DLBCL (Table 3).Moreover, we found a borderline significant association between sCD27 and all three BCL subtypes.Analyses using continuous measures of the markers showed similar results to the categorical analyses (Supplementary Table 3). ",
"section_name": "Association between immune markers and lymphoma development. Multivariable conditional",
"section_num": null
},
{
"section_content": "To explore the possibility of reverse causation, associations of the markers with risk of BCL and subtypes were further stratified by median (9 years) duration of time between blood donation and diagnosis of BCL.Unconditional logistic regression among subjects diagnosed ≤ 9 years from the time of blood collection essentially showed similar associations as presented in Table 2. Subjects diagnosed more than 9 years from the time of blood collection with elevated levels of sCD23 and sCD27 showed an increased risk of CLL, while increased levels of all markers were associated with higher risk of DLBCL (Supplementary Table 4).In the combined model (including all markers together), associations of sCD23 with CLL and CXCL13, sCD23, and sCD27 with DLBCL remained significant. Table 2. Multivariable conditional analyses: odds ratio (OR) and 95% confidence interval (CI) for individual immune marker (categorical variable) and B-cell lymphoma and histological subtypes.B-cell lymphoma (BCL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL); number of cases/controls (N); adjusted for BMI, education, smoking, alcohol intake, and physical activity; Quartiles were calculated based on the distribution in control subjects; tests for trend (p) were calculated using the quartile number as a continuous variable.Bold: statistically significant at p-value ≤ 0.05.The correlation between marker levels and time to diagnosis was evaluated.These analyses revealed higher levels of the markers in those cases with blood drawing closer to the diagnosis date (Fig. 1) except for sCD23 and sCD27 among DLBCL cases, which may suggest that serum levels of these markers are impacted by the disease itself. ",
"section_name": "Analyses stratified by time-to-diagnosis (TTD).",
"section_num": null
},
{
"section_content": "The AUC, as a measure of how well a marker (or a group of markers) predict the development of BCL subtype, was calculated by tenfold cross-validation.We found an AUC of 0.80 for a model including sCD23 as a predictor of CLL.Addition of other markers to the model did not significantly increase the prediction ability for CLL (Supplementary Table 5).sCD23 levels showed the highest prediction ability for CLL among male participants (AUC = 0.85, p = 0.0003) and more particularly those older than 60 years as indicated by an AUC 0.88 (p = 0.02) (Supplementary Table 6).sCD23 and CXCL13 showed a lower predictive ability for FL (AUC ~ 0.60) and DLBCL (AUC ~ 0.63) compared with CLL and addition of other markers did not significantly increase the AUCs for these subtypes (Supplementary Table 5). While the AUCs gave an overall picture of the behavior of the markers across all cutoff values, there remains a practical need to determine the specific cutoff value that could be used for individuals requiring screening.Therefore, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for different cut-off values of sCD23 for the prediction of CLL, FL, and DLBCL and cut-off values of CXCL13 for the prediction of FL and DLBCL were determined (Supplementary Table 78).Generally, there is a trade-off between sensitivity and specificity.Using the cut-off value showing the highest specificity, subjects with sCD23 levels ≥ 3,608 pg/mL appeared to have a 70% probability to develop CLL after 9 years (Fig. 2a, Supplementary Table 7), the probability being slightly higher for male (0.76) than for female (0.62) participants (Fig. 2b,c, Supplementary Table 7).When analyzing sCD23 and CXCL13 test accuracy for DLBCL and FL, it was observed that despite the specificity of sCD23 being the same for FL and DLBCL, PPVs were low compared to CLL (Supplementary Table 8). ",
"section_name": "Receiver operating characteristic (ROC) and test performance analysis.",
"section_num": null
},
{
"section_content": "Finally, to evaluate the hypothesis that immune markers act as a mediator on the causal pathway between known risk factors and B-cell lymphoma, a causal mediation analysis was conducted.Actual analyses were performed if (1) the risk factors were significantly associated with immune markers (Supplementary Table 9, model M) and (2) immune markers were found to be significantly associated with lymphoma subtypes in our combined models (Table 3).Selection of possible risk factors for mediation analyses was based on a large pooled study from 20 case-control studies 13 .Although, most of these associations were only suggestive and not significant in EPIC (Model X in Supplementary Table 9), the cohort can still help to Table 3.Odds ratio (OR) and 95% confidence interval (CI) for combined immune marker (categorical variables) and B-cell lymphoma and histological subtypes.B-cell lymphoma (BCL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL); adjusted for BMI, education, smoking, alcohol intake, and physical activity; to see the immune marker levels associated with each quartile see Table 2. Bold: statistically significant at p-value ≤ 0.05.understand potential mechanisms due to the prospective framework.Therefore, results of the mediation analysis should be seen as suggestive mediated associations.Positive association between BMI and DLBCL (average causal mediation effect (ACME) = 0.02) mediated through both sCD23 and CXCL13 (Table 4).We found a lower risk of DLBCL (ACME = -0.02)with physical activity mediated through CXCL13.Finally, there was a trend toward significance for a protective effect of alcohol intake (ACME = -0.05)mediated through sCD23 on CLL and for a protective effect of physical activity (ACME = -0.01)mediated through CXCL13 on FL (Table 4).Sensitivity analysis were conducted to evaluate the robustness of the results from the causal mediation analysis.The analyses showed that as long as ρ was 0.4 or lower, the estimated mediated effects still had the same sign, indicating good robustness. ",
"section_name": "Mediation analyses.",
"section_num": null
},
{
"section_content": "In this prospective study, we determined the serum levels of previously reported lymphoma associated immune markers and investigated how these markers correlate to the future risk of BCL histological subtypes and if risk of lymphoma is mediated by these markers.From these results, we confirmed the association between pre-diagnostically measured B-cell activation markers (sCD23, sCD27, sCD30, and CXCL13) and subsequent development of major BCL subtypes [8][9][10][11][12]28,29 . Afteradjustment for other immune markers, sCD23 remained significantly associated with all subtypes, while CXCL13 was found to be associated with FL and DLBCL only. The associations between sCD23 and CLL and DLBCL, and CXCL13 and DLBCL persisted among cases sampled more than 9 years before diagnosis, although the associations were attenuated in comparison to the findings from the models that covered the entire follow-up.sCD23 showed the highest prognostic value for CLL.In addition, we assessed for the first time, whether these markers may be mediating the causal pathway between several risk/protective factors and later lymphoma risk.Our results suggest that sCD23 and CXCL13 partly mediated the association between BMI (positive) and DLBCL risk, while lower levels of CXCL13 associated with higher physical activity and this partly explains the inverse association of physical activity and DLBCL risk.It should be noted that the measured biomarkers are individual markers in a complicated signaling milieu.Therefore any reported mediating effect for the marker itself should be interpreted as a proxy of the underlying biologic milieu that is captured by circulating levels of those molecules that is partly mediating the alleged associations.We observed strong associations between sCD23, sCD27, sCD30, and CXCL13 in blood samples collected up to 19 years before diagnosis and risk of BCL development, consistent with previous studies [8][9][10][11][12]28,29 . The asociations between sCD23 levels and development of BCL subtype were the most stable associations in this study, particularly for CLL, which was significant even more than 9 years before BCL diagnosis.In a recent study using the EPIC cohort with maximum 12.5 years TTD, ROC curve for the prediction or diagnosis of CLL indicates that sCD23 is a strong predictive marker (AUC = 0.88) 30 which is consistent with our current finding of AUC = 0.80.The earlier study included 179 CLL cases that were also present in our study.AUC based on new cases only (maximum 19 years follow-up) was 0.73.Moreover, our study showed that sCD23 is apparently more predictive in men than in women for future risk of CLL.Little is known how sex-specific factors influence CLL incidence.Although androgen receptors play a part in lymphopoiesis, it is unclear how this relates to the sex differences in CLL.As second explanation, sex-specific somatic alterations in the non-pseudoautosomal and pseudoautosomal regions on chromosomes X and Y have also been suggested to influence CLL incidence 30 . CD23 is an integral membrane glycoprotein involved in IgE binding, and is found on mature B-cells, activated macrophages, eosinophils, follicular dendritic cells, and platelets.CD23 is expressed on the membrane in CLL, in some cases of FL, and primary mediastinal large B-cell lymphoma.CLL cells have a characteristic phenotype of sIg low /CD19 + /CD5 + /CD23 +31 .CD23 has been widely used as a marker in the differential diagnosis of CLL versus mantle cell lymphoma.Its soluble form, sCD23, is released from activated B-cells and can itself induce further B-cell stimulation as well as function as a potent mitogenic growth factor.Many reports suggest that elevated CD23, either on neoplastic cell surfaces or as a soluble form, is a useful marker in either diagnosis or Table 4. Average direct effect and causal mediation effect (mediated through B-cell activation markers) of known risk factors on B-cell lymphoma subtypes.Diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL); Cases and control have been analyzed together; Correlation parameter (ρ) for mediated effect at which the sign of average causal mediation effect (ACME) is reversed; Effect estimates are the change in probability for moving the exposure variable from the reference category to the exposure category: alcohol intake: non-drinker (0) versus drinker (1), activity: first 3 categories (0) versus active(1), and BMI: < 30 (0) versus ≥ 30 (1); * \"average direct effect\" shows the estimate for unmediated effect which is the effect from any other path except the mediated one; † significant at p = 0.065; ‡ Shows the percentage of total effect mediated by the immune marker, it can be negative or greater than 100 depend on direct and mediated effect magnitudes and directions.Bold: statistically significant at p-value ≤ 0.05.prognosis of BCL 31 .Assuming that increased levels of sCD23 are caused by early stages of disease, our results further support that sCD23 would act as a marker for early detection of CLL 28 .In a study within the Northern Sweden Health and Disease Study (NSHDS), B-cell activation markers were measured in two pre-diagnostic blood samples donated by 170 individuals before BCL diagnosis, along with 170 matched cancer-free controls 29 .The study showed that regardless of baseline B-cell activation marker concentration, BCL future risk was also associated with an increase of marker concentrations over time (slope).The predictive ability of these markers for response to treatment as well as their prognostic value for disease progression in particular in the trajectory from monoclonal B-cell lymphocytosis (MBL), an asymptomatic condition in which small numbers of clonal B cells are detectable in blood, to CLL must be further assessed. CXCL13 is a CXC subtype member of the chemokine superfamily and acts via its receptor CXCR5 as one of the most potent B-cell chemo-attractants.CXCL13 expression is observed in BCL and diseases with B-cell activation.We reported that higher levels of CXCL13 were associated with increased risk of DLBCL, independently of sCD23, even among subjects diagnosed > 9 years after study initiation.Our finding is consistent with a previous report within the NSHDS (42 DLBCL cases) 29 , the Women's Health Initiative Observational Study cohort (138 DLBCL cases) 8 , and Nurses' Health Study and Health Professionals Follow-up Study (107 DLBCL cases) 32 .This would argue against the idea that increased marker concentrations would be attributable to undiagnosed disease, considering that the median survival of DLBCL is expected to be only a few months if left untreated 29 . The fact that measured B-cell activation marker levels were higher in those cases with blood drawing closer to the diagnosis date may indicate the existence of undiagnosed lymphoproliferative disease, in particular for indolent BCL subtypes.On the other hand, this may also reflect biological processes involved in the onset of the disease, e.g. as a measure of the allostatic load on the B-cell compartment related to non-heritable factors such as lifestyle and environmental factors [18][19][20][21][22][23][24][25] .Previous studies showed that elevated concentrations of these markers were associated with disease states related to immune system activation, such as autoimmune diseases, hepatitis C, and HIV infection [18][19][20][21][22][23] .However, such information was not available on our subjects, except for data on HBV infection from only a minority of the subjects (n = 335). Currently, no established screening programs for BCL development exist.Pre-diagnostic screening for risk factors of BCL in the general population has currently little clinical benefit.Moreover, cut-offs of marker levels to be used as a measure for disease progression have not been established yet.This would require the conduct of a large number of studies within various populations in order to establish normal levels and resulting in a more uniform reference for \"abnormal\" levels for use in clinical practice.In our view, the value of pre-diagnostic biomarkers is potentially much larger in groups of patients that are already at elevated risk of developing BCL, such as those with a family history of lymphoma and immunocompromised patients (i.e., primary immunodeficiency disorders, PID).PID patients carry an eightfold increased risk of lymphoma compared to the general population.Patients who have higher levels of the markers may thus be recommended to start intensive follow-up, while individuals with lower level of the markers may be advised for a less intensive follow-up.However, usefulness of these markers must be examined and validated among those patients before they could be rationally employed by physicians to improve human health. To our knowledge, this is the first study to assess the hypothesis of B-cell activation markers as mediators on the potential pathway between risk factors and later lymphoma development.BMI was associated with a significant increased risk of DLBCL mediated through sCD23 and CXCL13.There is no clear consensus on how obesity impacts B-cell development and function, but several studies point to an involvement of B-cells in adipose tissue inflammation associated with obesity [33][34][35] .A further study in obese and non-obese women revealed that body fat mass was positively correlated with total leukocyte, neutrophil, monocyte and lymphocyte counts 36 .While T-cell function was comparable between obese and non-obese women, B-cell function was about 50% higher in the obese group. Physical activity was associated with decreased risk of DLBCL and FL mediated through CXCL13.There has been evidence that moderate exercise is beneficial for the immune system 37 .Exercise induced changes in the number and function of cell subsets involved in the innate (e.g., neutrophils, monocytes, and natural killer cells) and the adaptive immune system (e.g., T and B cells) 37,38 .It has been shown that exercise can reduce insulin, glucose, and insulin-like growth factors, which may influence the proliferation of tumor cells in general.Physical activity also plays role in the prevention of obesity and reduces the percentage of body fat 38 .However, it is not clear if modulation of the immune system contributes to the potential antitumor properties of exercise. Adaptive immune responses, also called acquired immunity, is characterized by antigen-specific T-cell proliferation, immunological memory, B-cell activation, and production of antibodies.Evidence indicates that alcohol exposure can interfere with various aspects of the immune response and affect the different cellular components of the innate and/or adaptive immune system.Several studies reported that the number and function of B-cells are reduced by alcohol 26,27 .In our study, sCD23 mediated the association between alcohol intake and decreased risk of CLL.A meta-analysis provided evidence for a favorable role of alcohol drinking on NHL risk 39 .However, there is no clear biological explanation for this association. A strength of our study is the relatively large number of incident cases of newly diagnosed major BCL subtypes in a cohort of cancer-free individuals with pre-diagnostic blood samples and prospective follow-up times of up to 19 years.This enabled us to carry out specific analyses according to BCL subtypes.This is particularly relevant since there is growing evidence that lymphoma subtypes have different pathological and epidemiological features 13 .However, limitations of our study should be considered when interpreting the results.We measured blood immune markers at a single time point, which may not reflect accurately the long-term B-cell activation status and may not capture the most important etiologic timing.We cannot exclude potential measurement errors derived from dietary questionnaires, which could lead to systematic and random errors when estimating alcohol intake.Likewise, anthropometric measures were ascertained at recruitment (with the exception of the Oxford, France and Norway cohorts-self-reported).Moreover, we cannot exclude the possible bias due to unmeasured confounders (e.g., immune diseases and infections), which are well known risk factors of BCL.If an unmeasured confounder is related to several mediators this may have resulted in a bias in the mediation results 40 .Due to the limited sample size, the results of this sensitivity analysis should be interpreted with caution.Notably, the measured immune markers are not only produced by those cell types considered to play pivotal roles in the immune system (lymphocytes), but also by fibroblasts, neutrophils, eosinophils, follicular dendritic cells, and platelets.So, blood levels of the markers may not necessarily reflect activity in the target tissue (lymph nodes) 41 . In conclusion, increased B-cell activation marker levels present in blood years before BCL diagnosis, suggest a role of B-cell activation in BCL development at early stages.These may reflect a constitutional predisposition with shared underlying mechanisms for both indolent and aggressive lymphoma subtypes.Further studies investigating the biological and clinical impact of these markers are required.The mediating role of the immune function in the association between lifestyle factors and BCL also needs further examination. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Study population.The EPIC study is a prospective cohort study involving 23 centers from ten European countries (Denmark, France, Germany, Greece, the Netherlands, Italy, Norway, United Kingdom, Spain and Sweden) that was designed to investigate the potential relationships between diet, nutritional status, lifestyle and environmental factors and the incidence of cancer and other chronic diseases 42 .Over 500,000 healthy subjects in the age range 35-70 were recruited in the study during 1992-2000.The rationale, complete methodology and study design of the EPIC study have been described previously 42,43 .The ethical review boards from the International Agency for Research on Cancer and all local participating centers approved the study and all participants gave their informed consent.The study was conducted in accordance with the approved guidelines. Standardized lifestyle, medical and personal history, and diet questionnaires were collected from the participants, and a blood sample was taken at enrollment.Anthropometric measures were measured at recruitment (except for France, Oxford and Norway who collected self-reporting data).Within 2 h of blood collection, blood samples were processed for the isolation of buffy coats and other fractions.Samples were transported on dry ice to the laboratory and stored at -80 °C before analyses were performed. Incident lymphoma cancer cases were identified by population cancer registries for Denmark, Italy, The Netherlands, Norway, Spain, Sweden and the United Kingdom.A combination of methods was used in France, Germany and Greece, as detailed previously 42 .Originally, the diagnosis of lymphoma cases was based on the ICD-O-2.All lymphoma cases were subsequently reclassified according to the SEER ICD-O-3 morphology codes 44 .For each incident BCL case identified within the cohort by December 2012, one random control was selected among all cohort members alive and free of cancer at the time of diagnosis of the index case, matched by country, center, gender, age at recruitment (± 12 months), date of blood collection (± 3 months), time of blood collection (± 3 h), and fasting at blood collection. The current analysis was based on 516 case-control pairs for which a blood sample was available consisting of 174 DLBCL, 132 FL, and 210 CLL (including small lymphocytic leukemia) cases.For one CLL case and 9 controls, paired samples were missing.These subjects were included only in unconditional logistic analyses. Inclusion of etiological factors for FL, DLBCL, CLL subtypes.Previously known BCL risk and protective factors 13 that were available in EPIC were included in the study.These included BMI, height, smoking, education as proxy for SEP, alcohol intake and physical activity.The physical activity assessment included occupation as well as average recreational and household activity. Measurement of immune markers.Serum levels of sCD23, sCD27, sCD30, and CXCL13 were measured by ELISA for all samples (eBioscience, USA: BMS286INST, BMS240INST kits, and R&D Systems, USA: DCD230 and DCX130 kits).Assays were performed in duplicate and according to the manufacturers' protocols.All laboratory personnel were blinded with regard to case-control status.Matched case-control sets were assayed next to each other on the same plate in the same batch and quality control samples were run in duplicate along with the case-control sets in each batch.Inter-and intra-assay coefficients of variation were 2.2% and 3.1% for sCD23, 8.9% and 8.8% for sCD27, 4.8% and 7.3% for sCD30, and 5.2% and 6.8% for CXCL13. ",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "Marker levels measured out of range of the calibration curve (sCD23 = 6.3%, sCD30 = 0.8%, sCD27 = 0.5%, CXCL13 = 19.1%)and missing values of smoking status (2%), education (7%), alcohol intake (0.4%), physical activity (2.7%) covariates (Supplementary Table 10) were imputed based on a maximum likelihood estimation method which was informed by the observed correlation structure within the data 45 .Blood levels of soluble markers were log transformed to normalize their distributions.Differences between cases and controls in baseline continuous covariates were assessed using the paired t test, and by χ 2test, for categorical variables.Spearman rank correlation was used to measure the degree of correlation between markers. Odds ratios (OR) and 95% confidence intervals (95% CI) for the subtypes of BCL in relation to immune markers (as continuous variables) were calculated by conditional logistic regression (CLR).The models were adjusted for BMI (kg/m 2 , continuous), alcohol intake (g/day; continuous), smoking status (never, former, current), physical activity levels based on the Cambridge Physical Activity Index (inactive, moderately inactive, moderately active, active) 46 and educational level (none, primary, technical/professional, secondary, university/college).Quartiles of immune marker concentrations were calculated based on the distribution in control subjects, and CLR models were used to estimate the association between quartiles of marker levels and risk of BCL subtypes (first quartile as reference category).Tests for trend were calculated using the quartile number as a continuous variable.All markers were also modeled together (combined multivariable model) as it may be possible that one marker serves as a surrogate of another. Associations of the markers with risk of BCL and subtypes were additionally stratified by median duration of time between blood donation and diagnosis of BCL (time-to-diagnosis: TTD) to explore the possibility of reverse causation.In these analyses, to preserve statistical power, subtype cases were compared to all controls and (unconditional) models additionally adjusted for matching variables (i.e., country, gender, and age at recruitment) and plate number. Receiver operating characteristic (ROC) analysis and AUC comparisons were used to determine the discriminative ability of the markers separately or in combination with other markers.The AUCs was corrected for biases due to overfitting by tenfold cross-validation.For each fold, the AUC was calculated, and the mean of the fold AUCs was the cross-validated AUC estimate.Four objective measures of test performance were further calculated, namely, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for different cut-off values of the markers found to be informative for future risk of BCL 47 .Cut-off values were calculated based on deciles of ranked level of the markers in control subjects. Causal mediation analysis was applied to study the average causal mediation effect (ACME) and the average direct (unmediated) effect (ADE) of immune markers linking risk factors to lymphoma 48 .The effect estimates represent the change in probability that the subject develops lymphoma when moving the exposure variable from the reference category to the exposure category via the mediated or direct paths.Further, the analysis provides an estimate of the proportion of the total effect of exposure on lymphoma development mediated through the measured marker.Included exposure variables were smoking: non-smoker at recruitment (0) vs. smoker (1), alcohol intake: non-drinker (0) vs. drinker (1), physical activity: first 3 categories (0) vs. active(1), education: categories primary school or lower/ technical/ vocational school (0) vs. secondary school/ university/college (1), BMI: < 30 (0) versus ≥ 30 (1), and height: < country median (0) versus ≥ country median (1).We fitted two statistical models, the mediator (M) linear model for the conditional distribution of the mediator M given the risk factor X and a set of the covariates C; f(M | X, C), and the outcome (Y) logistic model for the conditional distribution of the outcome Y given X, M , and C; f(Y | X, M, C).These models were fitted separately and then their fitted objects comprised the main inputs to the mediate function, which computes the estimated ACME and other quantities of interest under these models and the sequential ignorability assumption.Mediation analyses were applied only for the risk factors significantly associated with immune markers and for the immune markers found to be significantly associated with lymphoma subtypes in our combined models (sCD23 and CXCL13).Adjustments were made for country, sex, and age.Models for each risk factor were additionally adjusted for other risk factors.There was no significant interaction between the risk factors and sCD23 and CXCL13.Quasi-Bayesian confidence intervals were determined 49 .Sensitivity analyses were performed for deviations from the sequential ignorability assumption (that in particular implies no unmeasured pre-sample collection confounders), with deviations measured by the correlation ρ between the errors in the mediation and the outcome models.In the presence of confounders which affect both the mediator and the outcome, we expect that the sequential ignorability assumption is violated and ρ is no longer zero 48 .A large critical ρ value reversing the sign of ACME indicates the violation of ignorability assumption 49 . Statistical analyses were performed using the R 3.4.1 language and environment (The R Foundation for Statistical Computing, Vienna, Austria) and SAS (version 9.4; SAS institute, USA).The R package mediation (4.1.2) was used for causal mediation analysis 49 .All p values are two-sided, with p < 0.05 considered as statistically significant. ",
"section_name": "Statistical analysis.",
"section_num": null
}
] |
[
{
"section_content": "The authors thank Dr. Marco W.J. Schreurs and the laboratory technicians Cindy Kneppers, Pamela Vasic, and Anneloes van Krimpen who performed the immune marker assays (Department of Immunology, Erasmus MC, University Medical Center, Rotterdam, the Netherlands), Moreover, the authors thank the National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands, for their contribution and ongoing support to the EPIC Study.European Commission (DG-SANCO), International Agency for Research on Cancer, Danish Cancer Society (Denmark), Ligue Contre le Cancer, Institut Gustave Roussy, Mutuelle Generale de l'Education Nationale, Institut National de la Sante et de la Recherche Medicale (INSERM) (France), German ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "The research protocol, data analysis plan, syntaxes, and analysis files can be available from the corresponding author.Data may not be shared as the EPIC explicitly retains ownership of the primary data and we agreed not to transfer the primary data disclosed under our agreement with the data owner to any third parties. ",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "F.S.H. contributed to concept and design of the research, data collection, laboratory work, statistical analysis and interpretation, and writing the article; P.M.K. contributed to the interpretation of the findings, critical revision of the article; D.C., A.N., M.S., S.N., P.F., J.D.M., E.W., and C.B. contributed to follow-up and/or management of the EPIC cohort, critical revision of the article; V.P. contributed to follow-up and/or management of the EPIC cohort, critical revision of the article and provided statistical expertise in mediation analysis; F.R.M., G.M., V.K., F.R., J.M.H., D.P., N.S., A.T., A.K., C.L.V., R.K., F.C., D.A., H.B., M.B.S., and A.P.-C.contributed to follow-up and/or management of the EPIC cohort, critical revision of the article; A.W.L. and V.H.J.V. contributed to the laboratory work, interpretation of the findings, critical revision of the article; R.V. contributed to follow-up and/ or management of the EPIC cohort, concept and design of the research, interpretation of the findings, critical revision of the article.All authors reviewed the manuscript. The authors declare no competing interests. ",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "F.S.H. contributed to concept and design of the research, data collection, laboratory work, statistical analysis and interpretation, and writing the article; P.M.K. contributed to the interpretation of the findings, critical revision of the article; D.C., A.N., M.S., S.N., P.F., J.D.M., E.W., and C.B. contributed to follow-up and/or management of the EPIC cohort, critical revision of the article; V.P. contributed to follow-up and/or management of the EPIC cohort, critical revision of the article and provided statistical expertise in mediation analysis; F.R.M., G.M., V.K., F.R., J.M.H., D.P., N.S., A.T., A.K., C.L.V., R.K., F.C., D.A., H.B., M.B.S., and A.P.-C.contributed to follow-up and/or management of the EPIC cohort, critical revision of the article; A.W.L. and V.H.J.V. contributed to the laboratory work, interpretation of the findings, critical revision of the article; R.V. contributed to follow-up and/ or management of the EPIC cohort, concept and design of the research, interpretation of the findings, critical revision of the article.All authors reviewed the manuscript. ",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "The authors declare no competing interests. ",
"section_name": "Competing interests",
"section_num": null
}
] |
10.7554/elife.79590
|
Notch signaling functions in non-canonical juxtacrine manner in platelets to amplify thrombogenicity
|
<jats:title>ABSTRACT</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>Notch signaling is an evolutionarily conserved pathway that dictates cell fate decisions in mammalian cells including megakaryocytes. Existence of functional Notch signaling in enucleate platelets that are generated as cytoplasmic buds from megakaryocytes still remains elusive.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>Platelets were isolated from human blood by differential centrifugation under informed consent. Expression of transcripts as well as peptides of Notch1 and DLL-4 in platelets was studied by employing RT-qPCR, Western analysis and flow cytometry. Platelet activation responses that include aggregation, secretion of granule contents and platelet-leucocyte interaction were analyzed by Born’s aggregometry, flow cytometry, Western analysis and lumi- aggregometry. Shedding of extracellular vesicles from platelets was documented with Nanoparticle Tracking Analyzer. Platelet adhesion and thrombus growth on immobilized matrix was quantified by employing microfluidics platform. Intracellular free calcium in Fura-2-loaded platelets was monitored from ratiometric fluorescence spectrophotometry. Coagulation parameters in whole blood were studied by thromboelastography. Ferric chloride-induced mesenteric arteriolar thrombosis in murine model was imaged by intravital microscopy.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Here we demonstrate significant expression of Notch1 and its ligand, the Delta-like ligand (DLL)- 4, as well as their respective transcripts, in human platelets. Synthesis and surface translocation of Notch1 and DLL-4 were upregulated when cells were challenged with physiological agonists like thrombin. DLL-4, in turn, instigated neighbouring platelets to switch to ‘activated’ phenotype, associated with cleavage of Notch receptor and generation of its intracellular domain (NICD). DLL-4-mediated pro-thrombotic attributes were averted by pharmacological inhibition of γ-secretase and phosphatidylinositol 3-kinase. Inhibition of Notch signaling, too, restrained agonist-induced platelet activation, and significantly impaired arterial thrombosis in mice, suggestive of synergism between thrombin- and DLL-4-mediated pathways. Strikingly, prevention of DLL-4-Notch1 interaction by a blocking antibody abolished platelet aggregation and extracellular vesicle shedding induced by thrombin.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>Our study presents compelling evidence in support of non-canonical Notch signaling that propagates in juxtacrine manner within platelet aggregates and synergizes with physiological agonists to generate occlusive intramural thrombi. Thus, targeting Notch signaling can be investigated as a potential anti-platelet/anti-thrombotic therapeutic approach.</jats:p></jats:sec><jats:sec><jats:title>Funding</jats:title><jats:p>This research was supported by J. C. Bose National Fellowship (JCB/2017/000029) and grants received by D. Dash from the Indian Council of Medical Research (ICMR) under CAR (71/4/2018-BMS/CAR), Department of Biotechnology (DBT) (BT/PR-20645/BRB/10/1541/2016) and Science and Engineering Research Board (SERB) (EMR/2015/000583), Government of India. S.N. Chaurasia is a recipient of financial assistance from the ICMR. M. Ekhlak is a recipient of CSIR-SRF and V. Singh is a recipient of UGC-SRF. D. Dash acknowledges assistance from the Humboldt Foundation, Germany. Funders have no role in the design, analysis and reporting of the study.</jats:p></jats:sec>
|
[
{
"section_content": "Notch signaling, one of the evolutionarily conserved pathways in mammals, is a central regulator of cell fate determinations through cell-to-cell interactions (Kopan and Ilagan, 2009;Guruharsha et al., 2012) that critically influences cell proliferation, differentiation and apoptosis (Miele and Osborne, 1999).Signaling is induced through binding of five independent ligands, Delta-like ligands (DLL)-1, 3, 4 and Jagged (Jag)-1 and -2 (Kopan and Ilagan, 2009), to four isoforms of cognate Notch receptors, Notch1 to Notch4, on surface of adjacent cells.Binding incites sequential α-and ϒ-secretasemediated proteolytic events releasing the intracellular domain of Notch receptor (NICD) that initiates downstream effects of Notch activation (Maillard et al., 2003;Blanpain et al., 2006;Qiao and Wong, 2009;Andersson et al., 2011). Platelets are circulating blood cells having central role in hemostasis and pathological thrombus formation that can lead to serious vaso-occlusive pathologies like myocardial infarction and ischemic stroke.Despite lack of genomic DNA platelets intriguingly express several transcription factors (Spinelli et al., 2010) and developmental morphogens like Wnt (Kumari and Dash, 2013;Steele et al., 2009) and Sonic Hedgehog (Kumari et al., 2014), whose non-canonical non-genomic roles in platelet biology and thrombogenesis remain poorly characterized.Notch signaling has been linked to differentiation of megakaryocytes (Sugimoto et al., 2006;Mercher et al., 2008), the platelet precursor cells in bone marrow, though there also have been reports to the contrary (Dorsch et al., 2002;Poirault-Chassac et al., 2010).Here, for the first time, we demonstrate abundant expression of Notch1 and DLL-4, as well as their respective transcripts in human platelets.When platelets were challenged with thrombin, a potent physiological agonist, synthesis and surface translocation of Notch1 and DLL-4 were significantly augmented.Interestingly, DLL-4, in turn, instigated activation of human platelets, as evidenced from binding of PAC-1 and fibrinogen to surface integrins α IIb β 3 , P-selectin externalization, release of adenine nucleotides, shedding of extracellular vesicles (EVs), amplified tyrosine phosphoproteome and rise in intracellular calcium, associated with generation of NICD.In parallel DLL-4 significantly enhanced phosphorylation of PI3K and AKT.Attenuation of γ-secretase significantly abrogated platelet activation responses triggered either by DLL-4 or thrombin.Inhibition of γ-secretase, too, significantly impaired arterial thrombosis in mice and platelet thrombus generation ex vivo.Furthermore, preclusion of DLL-4-Notch1 interaction by pre-incubation with a blocking antibody prohibited thrombin-mediated platelet aggregation and shedding of EVs, which underscores a critical role of Notch signaling in inducing human platelet activation in synergism with physiological agonists in a juxtacrine manner. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "No calculations were performed to predetermine sample size.Each experiment was performed independently at least three times or sample size was chosen based on effect size observed during pilot experiments.No inclusion and exclusion criteria were set for experimental units or data points.No outliers were excluded from the analysis. ",
"section_name": "Study design",
"section_num": null
},
{
"section_content": "Platelets were isolated from freshly drawn human blood by differential centrifugation.Briefly, peripheral venous blood collected in acid citrate dextrose (ACD) vial was centrifuged at 100×g for 20 min to obtain platelet-rich plasma (PRP).PRP was then centrifuged at 800×g for 7 min to sediment platelets after adding 1 µM PGE 1 and 2 mM EDTA.Pellet was washed with buffer A (20 mM HEPES, 134 mM NaCl, 2.9 mM KCl, 1 mM MgCl 2 , 0.34 mM NaH 2 PO 4 , 12 mM NaHCO3; pH 6.2) supplemented with 5 mM glucose, 0.35 g/dl BSA and 1 µM PGE 1 .Finally, platelets were resuspended in buffer B (20 mM HEPES, 134 mM NaCl, 2.9 mM KCl, 1 mM MgCl 2 , 0.34 mM NaH 2 PO 4 , 12 mM NaHCO3; pH 7.4) supplemented with 5 mM glucose.The final cell count was adjusted to 2-4×10 8 cells/ml using automated cell counter (Multisizer 4, Beckman Coulter).Leukocyte contamination in platelet preparation was found to be less than 0.015%.All steps were carried out under sterile conditions and precautions were taken to maintain the cells in resting condition.Blood samples were drawn from healthy adult human participants after obtaining written informed consent, strictly as per recommendations and as approved by the Institutional Ethical Committee of the Institute of Medical Sciences, Banaras Hindu University (Approval No. Dean/2015-16/EC/76).The study methodologies conformed to the standards set by the Declaration of Helsinki. ",
"section_name": "Platelet preparation",
"section_num": null
},
{
"section_content": "Washed human platelets were stirred (12,00 rpm) at 37 °C in a whole blood/optical lumi-aggregometer (Chrono-log model 700-2) for 1 min, followed by addition of agonist (thrombin, TRAP, or collagen) either in presence or absence of reagents.Aggregation was recorded as percent light transmitted through the sample as a function of time, while blank represented 100% light transmission.Platelet aggregation in whole blood, induced by either TRAP or collagen was recorded as change in electrical resistance (impedance) as a function of time. ",
"section_name": "Platelet aggregation",
"section_num": null
},
{
"section_content": "Proteins from platelet lysate were separated on 10% SDS-PAGE and electrophoretically transferred onto PVDF membranes by employing either a TE77 PWR semi dry blotter (GE Healthcare) at 0.8 mA/cm 2 for 1 hr 45 min or Trans-Blot Turbo Transfer System (Bio-Rad) at 20 V/1.3A for 30 min (for Notch1 and cleaved Notch1/NICD) or 20 min (for DLL-4, pY99, p-PI3K and pAKT).Membranes were blocked with either 5% skimmed milk or 5% bovine serum albumin in 10 mM Tris HCl, 150 mM NaCl, pH 8.0 containing 0.05% Tween 20 (TBST) for 1 hr at room temperature (RT) to block residual protein binding sites.Membranes were incubated overnight at 4 °C with specific primary antibodies, followed by 3 washings with TBST for 5 min each.Blots were incubated with HRP-conjugated secondary antibodies (goat-anti-rabbit, 1:2500 for anti-Notch1, anti-DLL-4, anti-cleaved Notch1, 1:1000, for p-PI3K, 1:1500 for anti-AKT and 1:40000 for anti-actin; and goat anti-mouse, 1:50000, for anti-pY99, 1:1500, for PI3K, 1:1000 for anti-pAKT) for 1 hr and 30 min at RT, followed by similar washing steps.Antibody binding was detected using enhanced chemiluminescence detection kit (Millipore).Membranes stained for p-PI3K and pAKT were subsequently stripped by incubating in stripping buffer at RT for 30 min, washed, blocked and reprobed employing either anti-PI3K or anti-AKT antibody.Images were acquired on multispectral imaging system (UVP BioSpectrum 800 Imaging System) and quantified using VisionWorks LS software (UVP). ",
"section_name": "Western analysis",
"section_num": null
},
{
"section_content": "Washed human platelets were stimulated with thrombin (1 U/ml) at 37 °C for 5 min under non-stirring condition.Cells were incubated with either anti-Notch1 antibody (1:100) for 1 hr at RT or anti-DLL-4 antibody (1:500) for 30 min at RT, followed by staining with Alexa Fluor 488-labelled anti-rabbit IgG (1:100, for Notch1; and 1:200, for DLL-4), for 30 min at RT in dark.Cells were washed, resuspended in sheath fluid and were analyzed on a flow cytometer (FACSCalibur, BD Biosciences).Forward and side scatter voltages were set at E00 and 350, respectively, with a threshold of 52 V.An amorphous gate was drawn to encompass platelets separate from noise and multi-platelet particles.All fluorescence data were collected using 4-quadrant logarithmic amplification for 10000 events in platelet gate from each sample and analyzed using CellQuest Pro Software. ",
"section_name": "Analysis of Notch1 and DLL-4 expression on platelet surface",
"section_num": null
},
{
"section_content": "Secretion from platelet α-granules in response to a stimulus was quantified by surface expression of P-selectin (CD62P).Washed human platelets pre-treated with either DAPT (10 µM) or vehicle for 10 min at RT followed by treatment with either DLL-4 (15 µg/ml) or DLL-1 (15 µg/ml) for 10 min at RT or thrombin (0.1 and 1 U/ml) for 5 min at 37 °C.In other experiments cell were pre-incubated with DLL-4 (7.5 µg/ml) followed by stimulation with thrombin (0.1 U/ml) for 5 min at 37 °C.Cells were stained with PE-labelled anti-CD62P antibody (5 % v/v) for 30 min at RT in dark.Samples were suspended in sheath fluid and subjected to flow cytometry.Secretion of adenine nucleotides from platelet dense granules was measured employing Chrono-lume reagent ( 0.2 μM luciferase/luciferin).Luminescence generated was monitored in a lumi-aggregometer contemporaneous with platelet aggregation (see above).Alternatively, dense granule releasate was quantitated using Cell Titer-Glo Luminescent Cell Viability Assay Kit where cells were sedimented at 800×g for 10 min and supernatant was incubated with equal volume of Cell Titer-Glo reagent for 10 min at RT. Luminescence was recorded in a multimodal microplate reader (BioTeK model Synergy H1). ",
"section_name": "Secretion from platelet α-granules and dense bodies",
"section_num": null
},
{
"section_content": "Platelet stimulation induces conformational switch in integrins α IIb β 3 that allows high-affinity binding of fibrinogen leading to cell-cell aggregate formation.Washed human platelets were pre-treated with either DAPT (10 µM), LY-294002 (80 µM) or Ro-31-8425 (20 µM) or vehicle for 10 min at RT followed by exposure to DLL-4 (15 µg/ml) or DLL-1 (15 µg/ml) for 10 min at RT or thrombin (0.1, 0.5 and 1 U/ml) for 5 min at 37 °C.In other experiments cells were pre-incubated with DLL-4 (7.5 µg/ml) followed by stimulation with thrombin (0.1 U/ml) for 5 min at 37 °C.Cells were stained with either FITC-labelled PAC-1 antibody that specifically recognizes active conformation of α IIb β 3 (5 % v/v) or Alexa Fluor 488-labelled fibrinogen (10 µg/ml) for 30 min at RT in dark.Samples were finally suspended in sheath fluid, and analyzed by flow cytometry. Isolation and analysis of platelet-derived extracellular vesicles (PEVs) PEVs were isolated and characterized as described previously (Chaurasia et al., 2019;Kushwaha et al., 2018).Platelets were pre-incubated either with DAPT (10 µM) or DBZ (10 µM) for 10 min, followed by treatment with DLL-4 (15 µg/ml) for 10 min at RT. Cells were sedimented at 800×g for 10 min, and then at 1200×g for 2 min at 22 °C ͦ to obtain PEVs cleared of platelets, which were analyzed with Nanoparticle Tracking Analyzer (NTA) where a beam from solid-state laser source (635 nm) was allowed to pass through the sample.Light scattered by rapidly moving particles in suspension in Brownian motion at RT was observed under 20 X microscope.This revealed hydrodynamic diameters of particles, calculated using Stokes Einstein equation, within range of 10 nm to 1 µm and concentration between 10 7 and 10 9 /ml.The average distance moved by each EV in x and y directions were captured with CCD camera (30 frames/s) attached to the microscope.Both capture and analysis were performed using NanoSight LM10 (Malvern) and NTA 2.3 analytical software, which provide an estimate of particle size and counts in sample. ",
"section_name": "Study of platelet integrin activation and fibrinogen binding",
"section_num": null
},
{
"section_content": "Intracellular calcium was measured as described (Chaurasia et al., 2019).Briefly, platelet-rich plasma (PRP) was isolated from fresh human blood and incubated with Fura-2 AM (2 µM) at 37 °C for 45 min in dark.Fura-2 labelled platelets were isolated, washed and finally resuspended in buffer B. Fluorescence for each sample was recorded in 400 µl aliquots of platelet suspensions at 37 °C under non-stirring condition by Hitachi fluorescence spectrophotometer (model F-2500).Excitation wavelengths were 340 and 380 nm and emission wavelength was set at 510 nm.Changes in intracellular free calcium concentration, [Ca 2+ ] i, was monitored from fluorescence ratio (340/380) using Intracellular Cation Measurement Program in FL Solutions software.F max was determined by lysing the cells with 40 µM digitonin in presence of saturating CaCl 2 .F min was determined by the addition of 2 mM EGTA.Intracellular free calcium was calibrated according to the derivation of Grynkiewicz et al., 1985. ",
"section_name": "Measurement of intracellular free calcium",
"section_num": null
},
{
"section_content": "Fresh human blood (20 µl) was added to a cocktail containing 10 µl each from APC-anti-CD41a (platelet-specific) and FITC-anti-CD14 (leukocyte-specific) antibodies and mixed gently.Samples were treated with either DAPT (40 µM) or vehicle for 10 min, followed by incubation with either TRAP (2 µM) or DLL-4 (15 µg/ml) for 15 min at RT. RBCs were lysed with 800 µl FACS lysis solution (1 X, BD Biosciences) for 10 min at RT. Platelet-leukocyte interaction was analyzed on a flow cytometer.Side scatter voltage was set at 350 with a threshold of 52 V and amorphous gates were drawn to encompass neutrophils and monocytes separate from noise.A dot plot of side scatter (SSC) versus log FITC-CD14 fluorescence was created in the CellQuest Pro software.Amorphous gates were drawn for monocyte (high fluorescence and low SSC) and neutrophil (low fluorescence and high SSC) populations.All fluorescence data were collected using 4-quadrant logarithmic amplification for 1000 events in either neutrophil or monocyte gate from each sample and analyzed using CellQuest Pro Software. ",
"section_name": "Study of platelet-leukocyte interaction",
"section_num": null
},
{
"section_content": "Coagulation parameters in whole blood were studied by employing Thromboelastograph 5000 Hemostasis Analyzer System (Haemonetics) and TEG analytical software.Whole blood (1 ml) was incubated either with DAPT (20 µM) or vehicle for 10 min at RT, followed by transfer to citrated kaolin tubes with proper mixing.CaCl 2 (20 µl) was added to 340 µl sample to initiate coagulation cascade.Mixture was placed in disposable TEG cups and data were collected as per to manufacturer instructions until maximum amplitude was reached or 60 min had elapsed. ",
"section_name": "Thromboelastography (TEG)",
"section_num": null
},
{
"section_content": "Ferric chloride-induced mesenteric arteriolar thrombosis in mice was imaged by intravital microscopy as previously described (Kulkarni et al., 2019;Chaurasia et al., 2019) with minor modifications.The animal study was ethically approved by the Central Animal Ethical Committee of Institute of Medical Sciences, Banaras Hindu University (Approval No. Dean/2017/CAEC/83).All efforts were made to minimize the number of animals used, and their suffering.Mice (species: Mus musculus; strain: Swiss albino; sex: male and female; age: 4-5 weeks old; weight: 8-10 g each) were anaesthetized with intraperitoneal injection of ketamine/xylazine cocktail (100 mg/kg ketamine and 10 mg/kg xylazine).Anti-GPIbβ antibody (DyLight 488-labelled, 0.1 µg/g body weight) diluted in 50 µl sterile PBS was injected into retro-orbital plexus of mice in order to fluorescently label circulating platelets.Mesentery was exposed through a mid-line incision in abdomen and kept moist by superfusion with warm (37 °C) sterile PBS.An epifluorescence inverted video microscope (Nikon model Eclipse Ti-E) equipped with monochrome CCD cooled camera was employed to image isolated mesenteric arterioles of diameter 100-150 µm.The arteriole was injured by topically placing a Whatman filter paper saturated with ferric chloride (10%) solution for 3 min and thrombosis in the injured vessel was monitored in real time for 40 min or until occlusion.Movies were subsequently analyzed with Nikon image analysis software (NIS Elements) to determine (a) the time required for formation of first thrombus (>20 µm in diameter), (b) time required for occlusion of the vessel i.e. time required after injury till stoppage of blood flow for 30 s, and (c) thrombus growth rate i.e. growth of a thrombus (>30 µm diameter) followed over a period of 3 min.Fold increase was calculated by dividing diameter of thrombus at given time (n) by the diameter of the same thrombus at time (0).Time 0 was defined as the time point at which thrombus diameter first reached the size 30 µm approximately. ",
"section_name": "Intravital imaging of thrombus formation in murine mesenteric arterioles",
"section_num": null
},
{
"section_content": "Platelet adhesion and thrombus growth on immobilized collagen matrix was quantified by using BioFlux (Fluxion Biosciences) microfluidics system as described previously (Sonkar et al., 2019).Wells of high-shear plates were coated with 50 µl collagen (from 100 µg/ml stock) at 10 dynes/cm 2 for 30 s and were left for 1 hr at RT. Wells were blocked with 1% bovine serum albumin at 10 dynes/cm 2 for 15 min at RT. Platelets stained with Calcein AM (2 µg/ml) were perfused over collagen at physiological arterial shear rate (1500 sec -1 ) for 5 min.Adhesion of platelets and thrombus formation in a fixed field over time was recorded.Representative images from 5 to 10 different fields were captured and total area occupied by thrombi at 5 min in 5 representative fields was analyzed using ImageJ software (National Institutes of Health). ",
"section_name": "Study of platelet thrombus formation on immobilized collagen matrix under arterial shear",
"section_num": null
},
{
"section_content": "Platelets were isolated from human blood as described above.Precaution was taken to prevent leukocyte contamination.Cells were counted with Beckman Coulter Counter Multisizer 4. Total RNA extraction, reverse transcription and qRT-PCR were carried out as described (Kumari et al., 2015). Total RNA was extracted from platelets using TRIzol reagent according to the protocol of the manufacturer and suspended in DEPC-treated water. ",
"section_name": "Quantitative real-time PCR RNA extraction",
"section_num": null
},
{
"section_content": "Platelet RNA (1 µg) was transcribed to cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the instructions of the manufacturer.Samples were amplified in a PTC-150 thermal cycler (MJ Research) by using the following program: 25 °C for 10 min, 37 °C for 2 hr, and 85 °C for 5 min. ",
"section_name": "Reverse transcription",
"section_num": null
},
{
"section_content": "Primers were designed using the latest version of Primer3 input software.The primers (forward and reverse) for target genes were obtained from Eurofins Genomics and presented in Table 1.Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and ACTB were used as the reference genes.We performed real-time PCR employing SYBR Green SuperMix in a CFX-96 real-time PCR system (Bio-Rad).Thermal cycling conditions were as follows: 95 °C for 3 min, followed by 40 cycles consisting of 10 s of denaturation at 95 °C, 10 s of annealing (at temperatures mentioned in the Table 1), and extension at 72 °C.A melt peak analysis of amplicons was carried out to rule out nonspecific amplifications. ",
"section_name": "Quantitative real-Time PCR",
"section_num": null
},
{
"section_content": "Standard statistical methods were employed in the study.Two tailed Student's t test (paired or unpaired) (for two groups) or RM one-way analysis of variance (ANOVA) (for more than two groups) with either Dunnett's or Sidak's multiple comparisons test was used for evaluation.Tests were considered significant at p<0.05.All the analysis was carried out employing GraphPad Prism version 8.4.Linear regression analysis was performed for in vivo studies, and the slopes from best-fit were used to arrive at rates in time-lapse experiments.Kalpan-Meier analysis and Log-Rank test were performed to determine significance of difference in time to occlusion of vessel between different groups.Data are presented as mean ± SEM of at least three individual experiments. ",
"section_name": "Statistical methods",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Although enucleate, platelets inherit a limited transcriptome from precursor megakaryocytes (Freedman, 2011;McRedmond et al., 2004).Notch1 is a transmembrane protein present on cell surfaces and is part of a highly conserved Notch signaling pathway (van Tetering et al., 2011).We searched for the expression of transcripts of Notch isoforms and its ligands in platelets by RT-qPCR.The Cq values for housekeeping genes (GAPDH and ACTB) were determined as 21 and 23, respectively, whereas that for NOTCH1 was 27 (Figure 1-figure supplement 1, A and B), which was reflective of abundant expression of NOTCH1 mRNA in human platelets.Contrasting this, NOTCH isoforms 2, 3 and 4 had Cq values greater than 33 (Figure 1-figure supplement 1, A and B).Keeping with above, there was notable existence of Notch1 peptide in human platelets, whose level significantly increased upon stimulation with thrombin (1 U/ml), a potent physiological agonist (Figure 1, A and B).Pre-treatment of platelets with puromycin (10 mM) singnificantly deterred synthesis of this pepetide (Figure 1, A and B).We also observed considerable expression of Notch1 on platelet surface membrane, whose level enhanced significantly (by 65.71%) upon thrombin-stimulation (Figure 1, C and D).The Cq of DLL4, JAG1, and JAG2 were found to be 26, 31 and 30, respectively while those for DLL isoforms-1 and -3 were higher than or equal to 33 (Figure 1 ).Consistent with above, platelets were found to express DLL-4 peptide whose level increased significantly when cells were challenged with thrombin (1 U/ ml) (Figure 1E andF).Rise in DLL-4 could be averted upon pre-incubation of platelets with puromycin (10 mM) (Figure 1E andF).Thrombin, too, significantly augmented surface translocation of DLL-4 by 64.31% (Figure 1G andH), thus raising possibility of DLL-4-Ntoch1 interaction on adjacent platelet membranes.As enucleate platelets are known to have limited capacity for protein synthesis, the present observations add Notch1 and DLL-4 to the growing list of platelets translatome. ",
"section_name": "Notch1 and DLL-4 are abundantly expressed in human platelets",
"section_num": null
},
{
"section_content": "Interaction of Notch1 with cognate ligands leads to sequential cleavage of the transmembrane receptor and generation of NICD (Iso et al., 2003).As Notch1 is expressed in human platelets, we asked whether exposure to DLL-4 would evoke release of NICD in these cells.Remarkably, exposure of platelets with DLL-4 (15 µg/ml) for 10 min led to significant rise (by 5.1-fold) in level of NICD (Figure 2A andB).As NICD generation is mediated through activity of γ-secretase, we next investigated the contribution of this protease in DLL-4-induced NICD release in platelets.Pre-treatment of platelets with either DAPT (10 μM) or DBZ (10 µM), specific inhibitors of γ-secretase, for 10 min led to significant drop in DLL-4-induced NICD release (by 25.33% and 23.77%, respectively) (Figure 2A andB), strongly suggestive of functional DLL-4-Notch1-NICD signaling axis in human platelets. Interestingly, level of NICD was reduced by 2.4, 43.4,70.3, and 84.9%, respectively, when platelets were stored for 1, 3, 5, and 8 hr at 37 °C in presence of 1 mM calcium (Figure 2C).However, NICD level was not considerably affected upon storage of cells at 22 °C.As calpain, the Ca 2+ -dependent thiol protease, is known to be activated in platelets stored at 37 °C, and not at 22 °C (Wadhawan et al., 2004), we pre-incubated cells at 37 °C with either calpeptin (80 µM) or ALLN (50 µM), specific inhibitors of calpain, or divalent ion chelator EGTA (1 mM).Significant recovery of NICD intensity under above conditions (Figure 2D) was consistent with NICD being a calpain substrate.In keeping with this observation, incubation of platelets with calcium ionophore A23187 (1 µM) for 10 min at 37 °C in presence of 1 mM calcium brought about significant reduction (by 29.77%) in the level of NICD, which was restored upon pre-treatment with either of the calpain inhibitors (Figure 2E).Hallmark of activated platelets is the conformational switch of its surface integrins α IIb β 3 that allows high-affinity binding of fibrinogen, associated with release of granule contents, rise in intracellular free calcium and shedding of extracellular vesicles.To study the effect of Notch ligands we pre-incubated platelets with DLL-4 (15 µg/ml for 10 min at RT) that prompted enhanced binding of PAC-1-FITC (that recognizes the open conformation of α IIb β 3 ) (Figure 3, A andB) and fibrinogen-Alexa Fluor 488 (Figure 3-figure supplement 1) by 3.07-and 3.13-folds, respectively.DLL-1 was notably ineffective in eliciting such response.Furthermore, platelets exposed to DLL-4 were found to have significant surface expression of P-selectin as a measure of α-granule secretion while no change was observed with DLL-1 (Figure 3, C and D).In keeping with above, DLL-4 also induced release of ATP from platelet dense granules (Figure 3E).Although DLL-4, on its own, did not incite platelet aggregation at the As Notch signaling is mediated through activity of γ-secretase leading to cleavage of Notch receptor, we next investigated the role of this protease in DLL-4-induced platelet activation.Platelets were pre-treated with DAPT (10 μM), a specific γ-secretase inhibitor, for 10 min at RT followed by exposure to DLL-4.Interestingly, we observed significant drop in DLL-4-induced activation of integrin α IIb β 3 (Figure 3, A and B; Figure 3-figure supplement 1), P-selectin exposure (Figure 3, C andD) and release of ATP from platelet dense granules (Figure 3E) when platelets were pre-incubated with DAPT. P-selectin expressed on stimulated platelets serves as a ligand for P-selectin glycoprotein ligand-1 (PSGL-1) receptor on leukocytes leading to platelet-leukocyte interaction.As DLL-4 incited P-selectin exposure on platelet surface, we asked next whether it would, too, prompt interaction between the two cell types.Remarkably, addition of DLL-4 (15 µg/ml, 10 min) to fresh human blood led to significant boost in platelet-neutrophil and platelet-monocyte aggregates, which was reduced upon pretreatment with DAPT (40 µM, 10 min) (Figure 3-figure supplement 2).Above observations underline a critical role of Notch signaling in platelet-leukocyte interaction and thrombogenesis. Rise in intracellular Ca 2+ , [Ca 2+ ] i , is a hallmark of stimulated platelets (Mallick et al., 2015).We next determined the possible effect of DLL-4 on calcium flux in human platelets.Interestingly, exposure to DLL-4 (15 µg/ml) for 10 min evoked significant rise (by 1.34-fold) in [Ca 2+ ] i in Fura-2 AM-stained platelets in presence of 1 mM extracellular Ca 2+ (Figure 3, F and G).To validate whether calcium entry from external medium contributed to rise in [Ca 2+ ] i , we pre-treated cells with EGTA (1 mM) followed by incubation with DLL-4.Chelation of extracellular calcium led to significant drop in rise in [Ca 2+ ] i (by 64.87%), suggestive of DLL-4-mediated Ca 2+ influx in these platelets (Figure 3, F and G). Platelet-derived extracellular vesicles (PEVs) are cellular fragments ranging in size between 0.1 and 1 µm that are shed by activated platelets (Kulkarni et al., 2019;Heijnen et al., 1999).PEVs are procoagulant in nature that significantly contribute to haemostatic responses (Sinauridze et al., 2007;Mallick et al., 2015).Exposure of platelets to DLL-4 (15 µg/ml) for 10 min led to extensive shedding of PEVs, which were 4.29-fold higher in count than those released from vehicle-treated counterparts (Figure 3H).Interestingly, pre-treatment of platelets with either DAPT (10 μM) or DBZ (10 µM), specific antibody (n=9) binding to platelets, respectively.(E) Bar diagram representing ATP secretion from platelet dense granules pre-incubated with either DAPT (10 µM) or vehicle for 10 min at RT followed by treatment with DLL-4 for 10 min (n=4).(F) Fura-2-loaded platelets were pre-treated for 5 min either with calcium (1 mM) or EGTA (1 mM) followed by incubation with DLL-4 (15 µg/ml) for 15 min and intracellular Ca 2+ was measured.(G) Corresponding bar diagram representing mean concentration of intracellular Ca 2+ over 300 sec of measurement (n=5).(H) Platelets were pre-treated with either DAPT (10 µM) or DBZ (10 µM) or vehicle for 10 min at RT followed by treatment with DLL-4 (15 µg/ml) for 10 min at RT. PEVs were isolated and analyzed with Nanoparticle Tracking Analyzer (n=8).(I) Immunoblot showing profile of tyrosine phosphorylated proteins in platelets pre-treated with either DAPT (10 µM) or DBZ (10 µM) or vehicle for 10 min at RT followed by treatment with either DLL-4 (15 µg/ml) for 10 min at RT or DLL-1 (15 µg/ml) for 10 min at RT or with thrombin (1 U/ml) for 5 min at 37 °C as indicated (n=4).Arrows indicate position of peptides whose intensity increased in presence of DLL-4.γ-secretase inhibitors, for 10 min led to significant drop in DLL-4-induced PEVs release (by 45.55% and 41.98%, respectively) (Figure 3H), thus underscoring critical role of γ-secretase activity. Platelet activation is associated with phosphorylation of multiple cytosolic proteins on tyrosine residues (Golden et al., 1990).Platelets treated with DLL-4 but not DLL-1 evoked increased tyrosine phosphorylation of peptides having Mr 72, 95, 110, and 124 kDa, which was remarkably reduced in presence of DAPT and DBZ.Above observations are indicative of DLL-4-γ-secretase axis-induced flux in tyrosine phosphoproteome in human platelets (Figure 3I).Thrombin-stimulated platelets were employed in the study as positive control. Roles of phosphatidylinositol (PI) 3-kinase and protein kinase C (PKC) in platelet activation have been widely reported (Hirsch et al., 2001;Polanowska-Grabowska and Gear, 1999;Atkinson et al., 2001;Watson and Hambleton, 1989).In order to implicate these kinases in DLL-4-mediated integrin α IIb β 3 activation, platelets were pre-treated with either LY-294002 (80 µM) or Ro-31-8425 (20 µM), inhibitors of PI3K and PKC, respectively, or vehicle for 10 min at RT, followed by incubation with DLL-4 (15 µg/ml) for 10 min.Strikingly, both the inhibitors triggered significant drop in DLL-4-induced PAC1 binding to platelets (Figure 3-figure supplement 3), which underscored the roles of PI3K and PKC in DLL-4-induced conformational changes in integrins α IIb β 3 .Consistent with activation of PI3K, phosphorylation of its p85 regulatory subunit was significantly augmented (by 3.85-fold) in presence of DLL-4 (15 µg/ml, 10 min), which, too, provoked significant upregulation (by 2.35-fold) in phosphorylation of AKT, the enzyme downstream of PI3K (Figure 3, J-M).The phosphorylations of PI3K as well as AKT were significantly attenuated by 30.05% and 36.67%,respectively, upon pre-treatment with DAPT (10 µM for 10 min) (Figure 3, J-M).These findings are strongly suggestive of non-canonical signaling evoked by DLL-4 in human platelets in γ-secretase-dependent manner leading to platelet activation. ",
"section_name": "DLL-4 amplifies expression of Notch intracellular domain (NICD) in human platelets",
"section_num": null
},
{
"section_content": "As thrombin triggers synthesis and expression of DLL-4 on platelet surface, which, in turn, induces platelet activation signaling, we asked next whether DLL-4 synergizes with thrombin in transforming platelets to 'pro-active / pro-thrombotic' phenotype.Interestingly, there was significant upregulation in platelet aggregation, PAC-1 binding and P-selectin externalization when cells were challenged with thrombin (0.1 U/ml) in presence of DLL-4 compared to samples exposed to thrombin alone (Figure 4, A-F).These parameters were considerably attenuated (by 50%, 30.34%, and 23.05%, respectively) upon prior exposure to DAPT (Figure 4, A-F).As Notch signaling is propagated through direct cellcell contact in juxtracrine manner, it is tempting to speculate that cellular proximity achieved within densely packed thrombus milieu would permit interactions between DLL-4 and Notch1 on surfaces of adjacent platelets that would synergize with physiological agonists in realizing thrombus consolidation. In order to implicate juxtracrine Notch signaling in amplification of platelet activity, we forestalled possible interaction between DLL-4 and Notch1 on adjacent cell surfaces by employing a rabbit polyclonal anti-DLL-4 antibody (20 µg/ml for 5 min) that would block DLL-4.In control samples a non-specific rabbit IgG (20 µg/ml) substituted the antibody against DLL-4.Remarkably, presence of followed by stimulation with thrombin (0.1 U/ml) as indicated.Tracings 4 of C and E represent cells pre-incubated with DAPT (10 µM) for 10 min at RT followed by addition of DLL-4 and thrombin.(D and F) corresponding mean fluorescence intensity of PAC-1 binding (n=9) and surface expression of P-selectin (n=10), respectively.(G) Aggregation of washed human platelets induced by thrombin (0.1 U/ml) following pre-treatment with either rabbit-IgG (20 µg/ml) for 5 min (tracing 2), or anti-DLL-4 antibody (20 µg/ml) for 5 min (tracing 3) or vehicle (tracing 1).(H) Corresponding bar chart representing mean platelet aggregation (n=4).(I) Platelets were pre-treated with either anti-DLL-4 antibody (20 µg/ml) or rabbit IgG (20 µg/ml) or vehicle for 5 min at RT followed by aggregation induced by thrombin (0.1 U/ml) for 5 min at 37°C.EVs were isolated from aggregated platelets and analyzed with Nanoparticle Tracking Analyzer (n=3).Data are representative of at least three different experiments and presented as mean ± SEM.Analyzed by RM one-way ANOVA with either Dunnett's multiple comparisons test (B, H, and I) or Sidak's multiple comparisons test (D and F).anti-DLL-4 antibody significantly impaired (by 81.95 %) platelet aggregation induced by thrombin (0.1 U/ml) compared with rabbit IgG-treated counterparts (Figure 4, G andH).The extent of drop in aggregation directly correlated with concentration of the blocking antibody in the range from 2 to 20 µg/ml (Figure 4-figure supplement 1).Furthermore, shedding of extracellular vesicles from aggregated platelets was also inhibited significantly (by 56.31%) when cells were pre-incubated with anti-DLL-4 antibody compared to rabbit IgG-treated control samples (Figure 4I).Above observations were strongly suggestive of juxtracrine Notch signaling operating within the confinement of tightly packed platelet aggregates / thrombi that potentiates platelet stimulation by thrombin. ",
"section_name": "DLL-4 operates in a juxtacrine manner to potentiate thrombin-mediated platelet activation",
"section_num": null
},
{
"section_content": "Thrombin and collagen are potent physiological agonists that elicit strong wave of platelet activation through their cognate receptors.Aggregation of washed human platelets induced by diverse agonists (thrombin, 0.25 U/ml; TRAP, 2.5 µM; or collagen, 2.5 µg/ml) were profoundly impaired (by 29.21, 20, and 71.8%, respectively) by DAPT (20 µM) (Figure 5, A-F), which, too, retarded TRAP, also known as PAR1-activating peptide (PAR1-AP) and collagen-mediated aggregation (by 46.88 and 28.97%, respectively) in whole blood analyzed from electronic impedance (Figure 5, G-J).Thrombin-induced ATP release from platelet dense granules was also attenuated when cells were pre-incubated with DAPT (Figure 5K).DBZ, another inhibitor of γ-secretase, also impaired thrombin-induced platelet aggregation (Figure 5-figure supplement 1).Interestingly, we also observed significant abrogation of thrombin-induced binding of PAC-1 (Figure 5-figure supplement 2, A-D) and fibrinogen (Figure 5-figure supplement 2, E and H) to platelet surface integrins, as well as decline in surface externalization of P-selectin (Figure 5-figure supplement 3, A-D) and shedding of extracellular vesicles (Figure 5-figure supplement 4), when cells were pre-incubated with DAPT (10 µM for 10 min at RT), which was suggestive of critical role of Notch signaling in amplification of agonist-stimulated platelet responses. Platelet interaction with circulating leukocytes is a sensitive index of state of platelet activity (Cerletti et al., 2012;Ortiz-Muñoz et al., 2014).In order to implicate Notch signaling in this, plateletneutrophil and platelet-monocyte aggregates were induced to form in whole blood with addition of TRAP (2 µM, 15 min).Strikingly, percent of cells undergoing aggregation were found to be significantly restrained upon pre-treatment with DAPT (40 µM, 10 min) (Figure 5, L-O), which further underlines a role of Notch signaling in platelet-leukocyte interaction and thrombogenesis. ",
"section_name": "Inhibition of γ-secretase attenuates agonist-induced platelet responses",
"section_num": null
},
{
"section_content": "Platelets play key role in the pathogenesis of arterial thrombosis.In order to implicate Notch signaling in generation of occlusive intramural thrombi in vivo, we studied the effect of pharmacological inhibitor of γ-secretase in a murine model of mesenteric arteriolar thrombosis.Platelets were fluorescently labelled and mice were intraperitoneally administered with either DAPT (50 mg/kg) or vehicle (control).Intramural thrombus was induced by topical application of ferric chloride in exteriorized mesenteric arterioles.Intravital imaging of thrombus was carried out by epifluorescence video microscope equipped with high-speed camera.We observed the time required for first thrombus formation, thrombus growth rate and time to occlusion as indicators to the initiation, propagation and stabilization of thrombus, respectively.Remarkably, mice administered with DAPT (Video 1) exhibited significantly delayed thrombus formation compared to vehicle-treated (Video 2) animals (mean times to form first thrombus: control, 4.25±1.52min; DAPT, 7.38±2.94min ) (Figure 6A andB).DAPT also impaired thrombus growth rate compared to vehicle-treated control counterparts (Figure 6C) (Videos 1 and 2).However, we did not observe significant difference in mean time to stable occlusion (Figure 6D).Kaplan-Meier analysis and log-rank test also showed no significant difference in occlusion times between control and DAPT-treated mice (Figure 6-figure supplement 1).Above observations attribute a critical role to platelet-specific γ-secretase in initiation and propagation of arterial thrombosis in vivo. Further, in order explore the role of Notch signaling in generation of thrombus ex vivo, we studied platelet dynamic adhesion and thrombus formation on immobilized collagen under physiological arterial shear (1500 s -1 ) employing BioFlux microfluidics platform.Washed human platelets were pretreated with either DAPT (20 µM) or vehicle (control) for 10 min at RT, and allowed to perfuse over the In keeping with above, we next analyzed the contribution of Notch signaling on intrinsic pathway of blood coagulation by employing kaolin-activated thromboelastography.Pre-treatment with DAPT (20 µM) significantly prolonged the reaction time (R) from 5.48±0.52-7.28±1.06min and attenuated maximum amplitude (MA) by 7.43% (Figure 6, G-I; Supplementary file 1), which was reflective of delayed formation of thrombus that was significantly less stable as compared with control counterparts with optimal γ-secretase activity.Thus, observations from the in vivo murine model of thrombosis as well as thromboelastography underscored an indispensable role of Notch pathway in determining thrombus stability. (tracing 2) of DAPT (40 µM) recorded as change in electrical resistance (impedance).B (n=7), D (n=5), F (n=3), H (n=3), and J (n=3), corresponding bar chart representing mean platelet aggregation.K, bar diagram representing mean ATP secretion from platelet dense granules (n=4).(L and N) Flow cytometric analysis of platelet-neutrophil aggregates (L) and platelet-monocyte aggregates (N) in whole blood stained with anti-CD41a-APC (specific for platelets) and anti-CD14-FITC (specific for neutrophils/monocytes) followed by treatment with TRAP (2 µM) in presence or absence of DAPT ( 40 ",
"section_name": "Inhibition of γ-secretase impairs arterial thrombosis in mice and platelet thrombus generation ex vivo",
"section_num": null
},
{
"section_content": "The Notch signaling has been implicated in production of megakaryocytes and platelets from CD34 + cells (Poirault-Chassac et al., 2010).However, expression of Notch receptors and its functionality in human platelets has remained unexplored.Notch signaling is mediated through four isoforms of mammalian Notch receptors, namely Notch1 to Notch4, which interact with five independent Notch ligands, DLL-1,-3, -4 and Jag-1 and -2 (Kopan and Ilagan, 2009).In this report we have demonstrated that, enucleate platelets have notable expression of Notch1 and its ligand DLL-4, which function in a non-canonical manner to synergize with physiological platelet agonists, leading to generation of prothrombotic phenotype.Although platelets have limited protein-synthesizing ability, exposure to thrombin instigated significant translation of DLL-4 and Notch1 in puromycin-sensitive manner, thus adding them to the growing repertoire of platelet translatome.Strikingly, thrombin, too, provoked translocation of these peptides to platelet surface membrane, raising possibility of juxtacrine DLL-4-Notch1 interaction within the confinement of platelet aggregates.DLL-4 stimulated significant rise in expression of NICD, the cleavage product of Notch, in a γ-secretase-dependent manner, that signifies the existence of functional DLL-4-Notch1-NICD signaling axis in platelets.This is not out of place here to mention that γ-secretase, too, is responsible for cleavage of amyloid-precursor proteins (APP) releasing amyloid-β (Aβ) (Tarassishin et al., 2004).Platelets, which contribute to 95% of circulating APP in body (Li et al., 1999;Davies et al., 1997;Bush et al., 1990), are known to generate Aβ 40 upon stimulation with physiological agonists like thrombin or collagen in a PKC-dependent manner (Smith and Broze, 1992;Skovronsky et al., 2001), leading to rise in local concentration of Aβ within the thrombus (Skovronsky et al., 2001;Smith, 1997). Platelets are central players in hemostasis and pathological thrombosis that can lead to occlusive cardiovascular pathologies like myocardial infarction and ischemic stroke.Upon activation platelet surface integrins α IIb β 3 switch to an open conformation, which allows high-affinity binding of fibrinogen and cell-cell aggregate formation, surface mobilization of P-selectin and rise in intracellular free calcium.In our quest to explore the non-genomic role of Notch pathway in platelet biology, we discovered that DLL-4 and not DLL-1 was able to instigate significant binding of PAC-1 (that recognizes the open conformation of α IIb β 3 ) and fibrinogen to platelets, associated with exocytosis of contents of alpha and dense granules, which was consistent with switch to a 'pro-active / pro-thrombotic' phenotype.DLL-4, too, provoked Ca 2+ influx leading to substantial rise in intracellular Ca 2+ , extracellular vesicle shedding, platelet-leukocyteaggregate formation and increase in platelet tyrosine phosphoproteome, all hallmarks of stimulated platelets.Notably, inhibition of γ-secretase employing two pharmacologically different compounds significantly impaired DLL-4-mediated 'pro-activating' effects on platelets.Inhibitors of either PI3K or protein kinase C evoked remarkable decrease in PAC1 binding, which underlines contributions of these enzymes in DLL-4-induced integrin activation.The phosphorylations of PI3K and AKT (downstream of PI3K) in platelets were significantly boosted in presence of DLL-4, which were attenuated upon pre-treatment with DAPT.In sum, above findings are strongly suggestive of non-canonical signaling triggered by DLL-4 in human platelets that operates in γ-secretase-PI3K-AKT-dependent manner leading to platelet activation. Thrombin is a potent physiological agonist that induces platelet aggregation and secretion through cognate PAR receptors.As thrombin amplifies expression of DLL-4 on platelet surface and DLL-4, in turn, induces platelet activation signaling, we asked whether DLL-4 synergizes with thrombin in Figure 6 continued stimulation of platelets.Pre-treatment of cells with DLL-4 followed by low dose (0.1 U/ml) thrombin significantly upregulated platelet aggregation, PAC-1 binding and P-selectin exposure elicited by thrombin alone, which underlines a potentiating effect by DLL-4.These responses were considerably abrogated by inhibitor of γ-secretase that further authenticates contribution of Notch signaling in platelet activation.Platelet aggregation induced by diverse agonists including thrombin, collagen and TRAP was also restrained by inhibiting γ-secretase activity.Remarkably, pharmacological inhibition of γ-secretase significantly impaired thrombus formation in a murine model of mesenteric arteriolar thrombosis and platelet thrombus generation ex vivo.As our laboratory and others have demonstrated Aβ to be a potent stimulus for platelets with thrombogenic attributes (Sonkar et al., 2014;Shen et al., 2008;Canobbio et al., 2014), pharmacologic inhibition of γ-secretase in platelets would prohibit release of both NICD and Aβ that may be envisaged as an effective multimodal anti-thrombotic strategy leading to thrombus destabilization.Kaolin-activated thromboelastography, too, validated delayed formation of thrombus that was significantly less stable compared with control counterpart having optimal γ-secretase activity.Taken together, above observations underscored seminal contribution from DLL-4-Notch1-γ-secretase axis in amplification of agonist-mediated platelet responses and determination of thrombus stability. As direct cell-cell contact is the mainstay of Notch signaling, it is reasonable to speculate interactions between DLL-4 and Notch1 on surfaces of adjacent platelets, which are closely approximated within the densely packed thrombus milieu.In order to validate it, we blocked proximity between DLL-4 and Notch1 by pre-incubating platelets with a rabbit polyclonal anti-DLL-4 antibody, followed by stimulation with thrombin.In control samples, a non-specific rabbit IgG substituted the antibody against DLL-4.Remarkably, presence of blocking antibody significantly impaired platelet aggregation evoked by thrombin compared with the rabbit IgG-treated counterparts.The extent of drop in aggregation directly correlated with concentration of the blocking antibody.In keeping with it, shedding of extracellular vesicles from aggregated platelets was also potentially inhibited when cells were preincubated with anti-DLL-4 antibody, and not with rabbit IgG.Above observations were overwhelmingly supportive of juxtracrine Notch signaling operating within the tightly packed platelet aggregate/ thrombus milieu that potentiates platelet stimulation by physiological agonists. In conclusion, we provide compelling evidence in favour of a PI3K /AKT-dependent non-canonical Notch signaling pathway operative in enucleate platelets that contributes significantly to the stability of occlusive arterial thrombus as well as to platelet activation instigated by thrombin through juxtacrine interactions (Figure 7).We demonstrate that inhibitors of γ-secretase, which downregulate Notch signaling in platelets, could be effective anti-platelet agents.Besides, antibody against DLL-4 may be employed therapeutically to forestall DLL-4-Notch1 interaction on surfaces of adjacent platelets as a potential anti-thrombotic approach. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "This research was supported by J C Bose National Fellowship (JCB/2017/000029) and grants received by D Dash from the Indian Council of Medical Research (ICMR) under CAR (71/4/2018-BMS/CAR), Department of Biotechnology (DBT) (BT/PR-20645/BRB/10/1541/2016) and Science and Engineering Research Board (SERB) (EMR/2015/000583), Government of India.SN Chaurasia, M Ekhlak and V Singh are recipients of ICMR-Scientist-C, CSIR-SRF and UGC-SRF support, respectively.D Dash acknowledges assistance from the Humboldt Foundation, Germany.The funders had no role in study design, data collection and interpretation, or decision to submit the work for publication. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Research was supported by grants received by DD from JC Bose Fellowship ",
"section_name": "",
"section_num": ""
},
{
"section_content": "All the data are available in the main text or the supporting information.Source data files have been provided for each figure included either in the manuscript or supplemental data. ",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "",
"section_name": "Additional information",
"section_num": null
}
] |
10.3389/fimmu.2022.943510
|
Interleukin-7 receptor signaling is crucial for enhancer-dependent TCRδ germline transcription mediated through STAT5 recruitment
|
<jats:p>γδ T cells play important roles in immune responses by rapidly producing large quantities of cytokines. Recently, γδ T cells have been found to be involved in tissue homeostatic regulation, playing roles in thermogenesis, bone regeneration and synaptic plasticity. Nonetheless, the mechanisms involved in γδ T-cell development, especially the regulation of TCRδ gene transcription, have not yet been clarified. Previous studies have established that NOTCH1 signaling plays an important role in the<jats:italic>Tcrg</jats:italic>and<jats:italic>Tcrd</jats:italic>germline transcriptional regulation induced by enhancer activation, which is mediated through the recruitment of RUNX1 and MYB. In addition, interleukin-7 signaling has been shown to be required for<jats:italic>Tcrg</jats:italic>germline transcription, VγJγ rearrangement and γδ T-lymphocyte generation as well as for promoting T-cell survival. In this study, we discovered that interleukin-7 is required for the activation of enhancer-dependent<jats:italic>Tcrd</jats:italic>germline transcription during thymocyte development. These results indicate that the activation of both<jats:italic>Tcrg</jats:italic>and<jats:italic>Tcrd</jats:italic>enhancers during γδ T-cell development in the thymus depends on the same NOTCH1- and interleukin-7-mediated signaling pathways. Understanding the regulation of the<jats:italic>Tcrd</jats:italic>enhancer during thymocyte development might lead to a better understanding of the enhancer-dependent mechanisms involved in the genomic instability and chromosomal translocations that cause leukemia.</jats:p>
|
[
{
"section_content": "",
"section_name": "GRAPHICAL ABSTRACT",
"section_num": null
},
{
"section_content": "gd T cells constitute a minor T-cell population (1-10% of all T lymphocytes) compared with canonical ab T cells (1).In addition to blood and secondary immune organs, where ab T cells reside, gd T cells accumulate in the gut mucosa, lung, skin, uterus, adipose tissue, meninges, liver and peritoneal cavity, playing important roles in the initiation and propagation of immune responses.During antigen recognition, gd T cells express a T-cell receptor (TCR), TCRgd, which can specifically respond to a variety of ligands, including nonpeptidic antigens, such as phosphoantigens and lipids that are not presented by major histocompatibility complex molecules, and peptides presented by the major histocompatibility complex (2,3).In addition, gd T-cell immune functions include (i) rapid production of large quantities of cytokines, (ii) killing of infected and tumor cells in a manner similar to natural killer cells, (iii) elimination of bacteria and other particles, and (iv) antigen presentation (1).Due to their innate and adaptive properties that enable them to robustly kill a wide range of tumor or infected cells, ability to present peptide antigens to ab T cells, and major histocompatibility complex-independent antigen recognition, increased interest has recently been directed to their potential use in novel immunotherapies (2).In addition, important roles played by gd T cells, including their functions in thermogenesis, bone regeneration, and synaptic plasticity, have been identified in tissue homeostasis (4)(5)(6)(7)(8).Despite the growing interest in these cells, the mechanism by which TCRd gene transcription is regulated during gd T-cell generation has not yet been clarified. During development in the thymus, T-cell precursors transition through a series of stages in which CD4 and CD8 are differentially expressed; these intermediates include CD4 - CD8 -double-negative (DN), CD4 + CD8 + double-positive (DP), and CD4 + or CD8 + single-positive (SP) thymocytes (9).Four DN populations, DN1 to DN4, are distinguished by the expression of CD25 and CD44; DN2 and DN3 thymocytes can be further classified into DN2a and DN2b and DN3a and DN3b, based on the expression of CD117 and CD27, respectively (9,10). Ordered expression of TCRgd and TCRab during thymocyte development is highly controlled to ensure the correct development of gd and ab T cells.TCRg and TCRd chains are simultaneously expressed in DN2b and DN3a thymocytes to generate TCRgd.The TCRb chain is expressed in DN3a thymocytes with an invariable pre-Ta chain, resulting in a TCR precursor known as pre-TCR, which induces cell proliferation, CD4 and CD8 expression leading to DP thymocyte generation, and TCRa chain expression.TCRa and TCRb chains are then simultaneously expressed in DP and SP thymocytes to form TCRab. Therefore, gd T cells arise from DN2b and DN3a thymocytes as a result of TCRgd expression, whereas ab T cells are derived from DP thymocytes as a result of TCRab expression.Because TCRg, TCRd and TCRb rearrangements occur in bipotent ab/gd T-cell precursors, the final outcomes derived from these events have an unquestionable impact on the ultimate T-cell fate (ab vs. gd Tcell), which is regulated by an instructive mechanism based on the stronger signaling of TCRgd than that mediated by the pre-TCR (11)(12)(13).Interestingly, pre-TCR signaling not only induces the expression of the TCRa chain but also induces the termination of TCRg and TCRd chain expression (14)(15)(16).Therefore, the exact control of the expression of these chains is crucial for the normal assembly of functional TCRs in thymocytes and the generation of gd and ab T cells (9,17). The ordered expression of the different TCR chains during thymocyte development depends on the specific regulation of enhancer-dependent germline transcription and V(D)J recombination at each individual TCR gene (9).These genes exist in two different conformations, unrearranged and rearranged, with a correctly rearranged configuration required for the expression of a functional chain (9).To pass from an unrearranged to a rearranged configuration, the enhancers present within the TCR genes play a critical role by triggering noncoding germline transcription initiated at the D and J gene segment promoters to promote accessibility of RAG proteins to the D-J region (18)(19)(20).V(D)J recombination-deficient mice, such as RAG-deficient mice, have a total block at the DN3a stage due to their inability to rearrange and express any of their TCR chains, as CD27 expression is dependent on intracellular TCRb expression (10,21).After rearrangement, transcription at the rearranged TCR genes depends on enhancer-dependent activation of the recombined V gene segment. Expression of the TCRg and TCRd chains depends on the activity of their respective transcriptional enhancers, Eg and Ed, which activate germline transcription of their unrearranged respective gene and subsequent recombination in DN2b to DN3a thymocytes (22,23).Successful VgJg and VdDdJd rearrangements (Figure S1) permit the expression of TCRgd in these cells, which drives thymocyte differentiation into gd T lymphocytes (10).Because Tcrg, Tcrd, and Tcrb germline transcription and recombination occur before TCRgd or pre-TCR expression in bipotent ab/gd T-cell precursors, these events are not directly involved in ab vs. gd T-lineage determination, which depends on the expression of TCRgd and pre-TCR (11)(12)(13).In DP thymocytes, Tcrg and Tcrd transcription is inactivated by pre-TCR signaling (14)(15)(16)23). Signaling mediated through NOTCH1 and interleukin-7 (IL-7) receptor (IL-7R) is essential for the generation of T cells (24).NOTCH1 signaling is indispensable for T-cell commitment at the DN2a thymocyte stage, and IL-7R signaling is required for thymocyte survival, proliferation and maturation and ultimately the generation of gd T cells (25)(26)(27)(28).Interestingly, the NOTCH1 and IL-7R signaling pathways constitute part of a transcriptional regulatory axis, in which IL-7Ra expression depends on NOTCH1 signaling (29)(30)(31).These signals are very strong in thymocytes from DN1 through the DN3a stages, decreasing abruptly during the transition to the DN3b thymocyte stage and in DP thymocytes due to inhibited NOTCH1 expression and subsequent reduction in IL-7Ra expression as a consequence of pre-TCR signaling (10,28,32). Previous experiments with DN3a thymocytes demonstrated that the activity of Eg and Ed measured by their ability to activate Tcrg and Tcrd germline transcription is induced by NOTCH1dependent recruitment of RUNX1 and MYB (14,16,(33)(34)(35)(36)(37); these factors are dissociated in DP thymocytes because of pre-TCR signaling-dependent inhibition of Notch1 expression, indicating a molecular mechanism of Tcrg and Tcrd silencing during thymocyte development (14).Hence, NOTCH1 plays an important role in enhancer-dependent Tcrg and Tcrd germline transcription and TCRgd expression during thymocyte development and thus in the generation of gd T cells.Interestingly, IL-7R signaling is required for Tcrg germline transcription and VgJg rearrangement (38)(39)(40)(41)(42)(43), explaining the absence of gd T lymphocytes in Il7ra -/-and Il7 -/-mice.IL-7Rdependent recruitment of STAT5 to Tcrg enhancers and promoters is essential for activating the noncoding germline transcription that triggers VgJg recombination (16, [44][45][46][47].STAT5 binding to Eg is lost in DP thymocytes because IL-7R signaling is terminated, constituting an additional mechanism of Tcrg silencing (16).In mature T cells, IL-7R signaling is also necessary for transcription of the rearranged Tcrg (48). Hence, both IL-7R-dependent STAT5 and NOTCH1dependent RUNX1 and MYB dissociation from Eg cause Tcrg silencing in DP thymocytes (14,16).Based on the parallel regulation of Eg and Ed by the NOTCH1/RUNX1 and MYB pathways in the regulation of Tcrg and Tcrd germline transcription during thymocyte development (14), we hypothesize that Ed also depends on the IL-7R/STAT5 pathway in DN3a thymocytes, similar to Eg.Our results demonstrate that IL-7R/STAT5 signaling is crucial for Eddependent Tcrd germline transcription.These data indicate that Ed and Eg are identically regulated through the same signaling pathways mediated by NOTCH1/RUNX1 and MYB and IL-7R/STAT5 in DN3a thymocytes, revealing indistinguishable mechanisms for expressing and silencing enhancer-dependent Tcrg and Tcrd germline transcripts during thymocyte development. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "Rag2 -/-and Il7ra -/-mice have been described previously (27,49).Three-to eight-week-old Rag2 -/-and Rag2 -/-x Il7ra -/-mice were used in this study.The animals were housed under pathogen-free conditions in the Animal Experimentation Unit at the IPBLN-CSIC in Granada, Spain, or the Institute for Frontier Life and Medical Sciences Resources in Kyoto, Japan.All animal work followed protocols approved by the CSIC and Andalusia Government Ethical Committees or the Kyoto University Animal Care and Use Committee. Cells and in vitro stimulations, inhibitions and viral transduction SCID.adh cells have been previously described (50).The cells used in this study were from the original parental cells, which are mostly committed to the T-cell lineage (51,52).They were cultured in RPMI 1640 medium with 10% fetal calf serum, sodium pyruvate, nonessential amino acids, glutamine, penicillin/streptomycin, and 50 mM 2-mercaptoethanol.Jurkat-green fluorescent protein (GFP)-and Jurkat-IL7Ra-GFP-expressing cells have been previously described (53).These cells were cultured in RPMI 1640 medium with 10% fetal calf serum, glutamine, and penicillin/streptomycin. SCID.adh cells (1 x 10 5 cells/mL) and Jurkat-GFP and Jurkat-IL7Ra-GFP cells (5 x 10 5 cells/mL) were stimulated in culture with 10 ng/mL murine recombinant IL-7 (Peprotech) for 30 minutes to 48 hours, as indicated.SCID.adh cells (1 x 10 5 cells/ mL) were incubated with 20 ng/mL phorbol acetate myristate and 0.5 mg/mL ionomycin (Sigma-Aldrich, Merck) or 16 mM gsecretase inhibitor 7(B-(-(3,5-difluorophenyl)-1-alanyl)-sphenyl-glycine t-butyl ester) (DAPT) (Selleckchem) for 24 hours.Viral transduction of SCID.adh cells with MigR retroviral plasmids was previously described (14,54). ",
"section_name": "Mice",
"section_num": null
},
{
"section_content": "To analyze transcription in SCID.adh cells, total RNA was obtained with peqGOLD TriFast (Peqlab) or Trifast (VWR).For RT-qPCR and the analysis of enhancer RNA (eRNA) transcripts in SCID.adh cells, genomic DNA-free RNA was obtained using Nucleospin plus columns (Macherey Nagel), and contaminating genomic DNA was eliminated by treatment with RNAse-free DNaseI (2270A, Takara) in the presence of an RNase inhibitor (2313A, Takara) for 1 hour at 37°C, followed by two consecutive phenol/chloroform extraction steps (Amresco/Merck).The DNase I treatment and extraction steps were repeated, and RNA was ultimately precipitated by adding ethanol to a final concentration of 70% with RNase-free glycogen as the carrier.The presence of genomic DNA contamination was determined by quantitative PCR (qPCR) using the Eg4 primers used in quantitative chromatin immunoprecipitation (qChIP) experiments.cDNA was obtained from 500 ng of total RNA with PrimeScript RT master mix (RR036, Takara) and dissolved in 100 mL with Milli-Q water.qPCRs were performed in 96-well plates (VWR) with 4 mL of cDNA in 10-mL reactions prepared in duplicate using TB Green Premix Ex Taq II (RR820, Takara) on a Bio-Rad CFX-96 System.The qPCR conditions were 95°C for 7 minutes, 40 cycles of 95°C for 30 seconds, 59.5°C for 45 seconds, and 72°C for 30 seconds, followed by incubation at 95°C for 1 minute.To analyze transcription in mouse thymocytes, qPCR was performed in 96-well plates using 1 mL of cDNA and 0.24 mL of 50 X ROX in 12 mL reactions in duplicate using TB Green Premix Ex Taq II (RR820, Takara) on a StepOnePlus qPCR machine (Applied Biosystems).The qPCR conditions were 40 cycles of 95°C for 30 seconds and 59.5°C for 30 seconds, followed by incubation at 95°C for 1 minute.Melting curve analyses were performed with 55°C -90°C ramping in 0.5°C steps and 5-second increments to confirm a single amplicon for each sample and primer pair analyzed.The expression of individual genes was calculated using the DCt method and normalized to Actb transcription.All RT-qPCR experiments were performed with at least three biological replicates.The primers for Actb, ACTB, Cg and Cd transcripts have been previously described (14).The primers were obtained from Metabion and Integrated DNA Technologies, and their sequences are listed in Table S1.Primer sequences for eRNA detection are shown in Table S1 and Figure S2. Analyses of assays for transposaseaccessible chromatin using sequencing (ATAC-seq), chromatin immunoprecipitation using sequencing (ChIP-seq), and transcriptome (RNA-seq) databases Guidelines for the design of primers for detection of eRNAs based on factor binding detection by ChIP-seq were previously described (55).To design the primers to detect eRNAs, we focused our search on the 250-500 bp sequences flanking the 324-bp mouse Ed fragment, based on its homology with the equivalent human Ed fragment, and the 227-bp mouse Eg4 fragment, where functional transcription factors are known to bind (44, 56) (Figures S2,S3).To confirm that the designed primers are specific for detecting Ed and Eg4 transcripts, we analyzed chromatin profiles, transcript annotation, candidate cis-regulatory elements (cCREs), factor binding by ChIP-seq and RNA-seq in a 2.6-kb Ed region and a 2.8-kb Eg4 region using available databases (Figures S2, S4-S6).ATAC-seq data in DN2b and DN3 thymocytes and gd T lymphocytes were retrieved from the Immunological Genome Project databrowsers (www.immgen.org)(57).Transcript annotation from GENCODE and the National Center for Biotechnology Information, cCREs from the ENCODE Registry, and transcription factor ChIP-seq information from ReMap Atlas of Regulatory Regions were retrieved using the UCSC Genome Browser.The ENCODE Registry of cCREs includes DNAseI hypersensitive sites across ENCODE samples that are supported by eH3K4me3, H3K27ac or CTCF binding by ChIP-seq.RNA-seq and H3K27ac ChIPseq analyses in DN thymocytes were obtained from data series GSE80272 (58) and analyzed using Integrative Genomic Viewer (https://igv.org)(Figure S6). ",
"section_name": "Quantitative reverse transcription polymerase chain reaction",
"section_num": null
},
{
"section_content": "For use in EMSAs, SCID.adh cell extracts were obtained from 10 7 unstimulated and mouse recombinant IL-7-stimulated cells for 30 minutes at 37°C.After washing with Hank´s balanced salt solution (Cultek), cells were resuspended in 200 mM NaCl, 50 mM Tris-HCl (pH: 8.0), 0.75 mM spermidine, 0.15 mM spermine, 0.1 mM EDTA, 0.1 mM Na 3 VO 4 , 1 mM DTT, 0.5 mM PMSF, and 1X complete protease inhibitors (Roche, Merck), lysed by adding Nonidet-40 to a 10% solution to a final concentration of 0.4% and incubated for 30 minutes on ice.Lysates were clarified by centrifugation at 12,000 × g for 10 minutes at 4°C, and glycerol was added to a final proportion of 25%.The protein concentration was determined by the Bradford assay (Bio-Rad).A total of 60,000 cpm of 32 P-labeled doublestranded oligonucleotide was incubated with 12 mg of cell extract in a 25-mL volume containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 2% glycerol, 1 mg of poly(dI-dC), and 1 mg of bovine serum albumin for 20 minutes on ice.One microgram of anti-STAT5 antibody (Santa Cruz Biotechnology, sc-835), which recognizes STAT5a and STAT5b, was added and incubated for 30 minutes at room temperature to supershift the specific complex.The binding sites are listed in Table S1.Native polyacrylamide (4.5%) containing bis-acrylamide/ acrylamide (1:19) containing 0.25X Tris-borate-EDTA previously run at 200 V for 1 hour was used to separate the DNA and DNA/protein complexes.The gels were fixed with 30% methanol and 10% acetic acid for 30 minutes and then dried and exposed to film.The primers of the tested binding sites were obtained from Metabion, and the sequences are listed in Table S1. ",
"section_name": "Electrophoretic mobility shift assays",
"section_num": null
},
{
"section_content": "qChIP experiments were performed with chromatin from 10 7 cells incubated with 5 mg of anti-STAT5 (Santa Cruz Biotechnology, sc-235), trimethylated lysine 4 of histone H3 (H3K4me3) (ab8580, Abcam), acetylated lysine 27 of histone H3 (H3K27ac) (ab4779, Abcam), or control (clone 1-1, Millipore, Merck or ab46540, Abcam) antibodies as previously described (14).The primers used for Eg, Ed, the Tcra enhancer (Ea), and Oct2 exon in the qChIP have been previously described (14,59).The primers were obtained from Metabion, and the sequences are listed in Table S1. ",
"section_name": "Quantitative chromatin immunoprecipitation",
"section_num": null
},
{
"section_content": "Reporter plasmids containing the firefly luciferase reporter gene driven only by a human TRDV1 promoter (Vd1p) alone and a human 370-bp Ed fragment driven by Vd1p were constructed based on the pXPG plasmid as previously described (60).Reporter plasmids containing the firefly luciferase reporter gene driven only by a minimal murine Fos promoter (cfosp) and by cfosp with murine 410-bp Eg1 were constructed based on the pGL4.10 plasmid (Promega) as previously described (14,44).To introduce a point mutation in the STAT5-binding site present in the dE6/7 region (dE6/7) of the Ed370-Vd1p-luciferase plasmid, a Q5 site-directed mutagenesis kit (E0554, New England Biolabs) was used with HPLC purified primers designed by the NEBaseChanger program.The sequences of the primers used are listed in Table S1.The mutation was confirmed by DraI digestion and sequencing.For luciferase assays, 5x10 6 Jurkat-GFP or Jurkat-IL7Ra cells were transfected by electroporation with 5 mg of the firefly luciferase reporter plasmid and 10 ng of the pRL-TK (Promega) Renilla luciferase reporter plasmid.Both electroporation and measurements of firefly and Renilla luciferase activities were performed as previously described (14). ",
"section_name": "Luciferase assays",
"section_num": null
},
{
"section_content": "Statistical analysis was performed with Prism 5.0 software (GraphPad).At least three independent experiments were performed in all cases.The number of independent experiments analyzed (n) is indicated in the figure legends.Nonparametric unpaired Student´s t tests with the Welch correction were performed, and significant differences between the indicated values are indicated by asterisks as follows: p <0.05 (*), p < 0.005 (**), and p<0.0005 (***).The absence of an asterisk indicates that the change relative to the control was not statistically significant. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Tcrd is flanked by Tcra Va and Ja gene segments and comprises Vd gene segments interspaced with Va segments within an ~1 Mb region, followed by a 33.7-kb region that contains two Dd (Trdd1 and Trdd2) gene segments, two Jd (Trdj1 and Trdj2) gene segments, Ed, the Tcrd C region (Cd), and the inverted Trdv5 gene segment in murine chromosome 14 (9) (Figure S1).Tcrg spans 0.2 Mb and comprises three functional Vg-Jg-Tcrg C region (Cg1, Cg2 or Cg4)-Eg clusters in murine chromosome 13 (9) (Figure S1).Expression of the TCRg and TCRd chains results from the activation of enhancerdependent germline transcription of their respective unrearranged genes, which induces long-range chromatin changes that trigger VgJg and VdDdJd recombination in DN2b and DN3a thymocytes (Figure S1).These noncoding transcripts are initiated by promoters associated with the Jg, Dd, and Jd gene segments that are ultimately spliced into their respective constant regions (40, 61) (Figure 1).The levels of germline transcription measured at these constant regions represent the sum of all the transcripts that are initiated in the D and/or J gene segments in their respective gene or gene cluster.To evaluate the potential role played by IL-7R signaling in the activation of Tcrd germline transcription, cells of the appropriate developmental stage that are deficient in V(D)J recombination must be used.We analyzed the levels of Cd transcripts in untreated and IL-7treated SCID.adh cells and compared them with the well-known regulation of Tcrg germline transcription by measuring IL-7dependent activation of Cg transcripts (40, 41) (Figure 2A).These cells, which were derived from mice carrying an inactivating spontaneous point mutation in the catalytic subunit of DNA-PK, exhibit a DN3a-like phenotype derived from their complete defect in V(D)J recombination (50, 62).Because their TCR genes are in a germline unrearranged configuration, these cells constitute an excellent model with which to study IL-7Rdependent Tcrg transcription, as well as pre-TCR-induced silencing of Tcrg and Tcrd and activation of Tcra (14,16,44,45,60,63).Due to a deletion at the 5´-end of the Tcrg locus in these cells, germline transcription of Cg4 (Cg) was analyzed as representative of the three Vg-Jg-Cg clusters because they share the same regulation (16) (Figure 1).Although basal Cd transcription was found to be higher than Cg transcription in these cells, IL-7 treatment clearly induced both Cg and Cd transcription (Figures 2A,B). ",
"section_name": "IL-7R signaling activates Tcrd germline transcription in DN3a thymocytes",
"section_num": null
},
{
"section_content": "Regulation of enhancer-dependent Tcrg and Tcrd germline transcription is regulated by Notch signaling (14).Because IL-7Ra is a target of Notch signaling (14,(29)(30)(31), we evaluated the effect of gain and loss of NOTCH1 signaling on Il7ra-and IL-7dependent Cg and Cd transcription (Figure 3).As expected (14), transduction of SCID.adh cells with intracellular NOTCH1 domain (ICN1)-expressing retroviruses induced Il7ra transcription (Figure 3A).Accordingly, IL-7-dependent activation of Cg and Cd transcription was induced in SCID.adh cells that had been transduced with ICN1 + GFPexpressing retroviruses, and the transcription levels were compared with those of cells that had been transduced with retroviruses that expressed only GFP (Figures 3B,C).In contrast, cell treatment with the g-secretase inhibitor DAPT, which inhibits proteolytic cleavage and thus prevents the release of endogenous ICN1, inhibited Il7ra transcription (Figure 3D); therefore, a decrease in IL-7-dependent activation of Cg and Cd transcription was detected (Figures 3E,F mechanism for the regulation of Tcrg and Tcrd germline transcription by this regulatory axis is based on the regulation of IL-7R expression by Notch signaling, which results in increased responsiveness of the unrearranged Tcrg and Tcrd genes to IL-7. ",
"section_name": "IL-7R-dependent activation of Tcrg and Tcrd germline transcription is regulated by Notch signaling",
"section_num": null
},
{
"section_content": "To study TCR germline transcription, thymocytes of the appropriate developmental stage (such as DN3a in the case of Tcrg and Tcrd) that are deficient in V(D)J recombination must be analyzed.Rag2 -/-mice show deficient V(D)J recombination; therefore, thymocyte development is blocked at the DN3a stage in these mice (21,49,64).In fact, these animals constitute a pure source of DN3a thymocytes, 99.0 ± 0.8% of total thymocytes (64).The Tcrg and Tcrd in an unrearranged configuration in these mice allowed us to analyze germline transcription in DN3a thymocytes.To clearly determine the role played by IL-7R signaling in vivo, we compared Cg and Cd germline transcription in Rag2 -/-and Rag2 -/-Il7ra -/-DN3a thymocytes by performing RT-qPCR (Figure 4A).Because Rag2 -/- thymocyte blockade occurs earlier during development and predominates over Il7ra deficiency (28,49), both Rag2 -/-and Rag2 -/-Il7ra -/-mice have an equivalent block at the DN3a stage. As expected (40), Cg transcription was abrogated in Rag2 -/- Il7ra -/-DN3a thymocytes.Our analyses of Cd transcription indicated that Tcrd germline transcription was also strongly dependent on IL-7R signaling (Figure 4A).Cd transcripts constitute the sum of Tcrd germline transcripts initiated at the Trdd2, Trdj1, and Trdj2 promoters (Figure 1).We also analyzed specific transcripts initiated at each of these promoters.The transcripts initiated at the Trdj1 and Trdj2 promoters are spliced to the first exon of Cd, while those initiated at the Trdd2 promoter are first spliced to the Trdj1 gene segment before splicing to the Cd first exon (Figure 1).The Trdd2-Trdj1, Trdj1-Cd and Trdj2-Cd transcripts were clearly detected in Rag2 -/- thymocytes (Figure 4B).According to the strong inhibition of Cd transcription, the aforementioned transcripts were profoundly inhibited in Rag2 -/-Il7ra -/-thymocytes (Figure 4B).These results indicate that, similar to Tcrg germline transcription, Tcrd germline transcription depends on IL-7R signaling in DN3a thymocytes. ",
"section_name": "IL-7R signaling is essential for Tcrd germline transcription in vivo",
"section_num": null
},
{
"section_content": "IL-7R signaling results in rapid phosphorylation of STAT5, which is translocated from the cytoplasm to the nucleus to activate its target genes.Accordingly, IL-7R signaling activates Cg transcription through the recruitment of STAT5 to Eg (44).We compared STAT5 binding to Eg4 and Ed by qChIP in unstimulated and IL-7-stimulated SCID.adh cells after a 30minute treatment (Figure 5A).We found comparable STAT5 recruitment to both Eg4 and Ed upon IL-7 treatment.To confirm the recruitment of STAT5 in primary DN3a cells, we evaluated its binding in Rag2 -/-thymocytes (Figure 5B).IL-7 treatment was not necessary to detect STAT5 binding to these enhancers in ex-vivo Rag2 -/-thymocytes, most likely because these cells were already stimulated in vivo.We found similar STAT5 binding to both enhancers, confirming the results obtained with SCID.adh cells.As a negative control in our qChIP experiments, STAT5 binding to an Oct2 exon sequence was also analyzed (Figures 5A,B). IL-7R signaling activates Ed function through STAT5 binding to the dE6/7 site eRNAs together with epigenetic activation marks on histone H3, such as trimethylation of lysine 4 (H3K4me3) and acetylation of lysine 27 (H3K27ac) are predictors of enhancer activity (65)(66)(67)(68).To evaluate whether IL-7 treatment can directly activate Ed and Eg activity, we analyzed the effect of IL-7R signaling on H3K4me3 and H3K27ac on Ed and Eg4 in unstimulated and IL-7-treated SCID.adh cells (Figures 6A,B).Consistent with the presence of these chromatin marks on active enhancers (67,68), we found that H3K4me3 and H3K27ac modification was strongly induced at both enhancers, but not at a negative control sequence, after IL-7 stimulation of SCID.adh cells.Detection of eRNAs is the most reliable indicator of enhancer activity (65,66).These noncoding transcripts are unidirectional or bidirectional and have low abundance due to their instability.Enhancer activation correlated with IL-7-dependent induction of bidirectional Ed and Eg4 eRNAs in SCID.adh cells (Figures 6C,D).To examine the presence of other cis-regulatory regions in the surrounding enhancer regions, we analyzed chromatin accessibility in DN2b and DN3 thymocytes, and gd T lymphocytes by ATAC-seq using the Immunological Genome Project databrowsers (www.immgen.org)(57), as well as the presence of other enhancers in the vicinity according to the ENCODE Registry of cCREs (Figures S2,S4,S5).Although these analyses indicate the presence of other cis-regulatory elements located in the vicinity of Ed and Eg4 within a region of less than 2 kb, these enhancers constitute the sequences with the highest density of transcription factor binding by ChIP-seq according to ReMap Atlas of regulatory regions (Figures S4,S5).Interestingly, p300 and STAT5 are specifically recruited to these enhancers (Figures S4,S5).Taken together, these data clearly demonstrate that Eg and Ed are both activated by IL-7R signaling. To directly evaluate the role of IL-7 on Ed function, we analyzed its effect on enhancer activity using luciferase reporter constructs in transiently transfected Jurkat cells (Figure 7).These cells constitute a well-established model for studying TCR enhancer activity upon cell stimulation (14,60).Because these cells express very low levels of IL7Ra, we used two Jurkat clones that had been previously obtained through retroviral transduction and that expressed GFP or IL7Ra + GFP (53).As shown in Figure 7A, the GFP-expressing cells exhibited very low levels of IL7RA expression compared with the cells transduced with GFP + IL7Ra-expressing retroviruses.As expected, Eg activity was highly activated by IL-7 treatment in the IL7Ra + GFP-expressing cells but not in the control GFP-expressing cells (Figure 7B).Similarly, we found that Ed activity was activated by IL-7 only in the IL7Ra + GFP-expressing cells and not in the control GFP-expressing cells (Figure 7B).The observed effects were clearly mediated by the respective enhancer because the luciferase activity of the constructs with no enhancer in either clone was unaffected by IL-7 treatment.Of the two conserved putative STAT5 sites found by comparing murine and human Ed sequences (Figure S3), we validated by EMSA the STAT5binding site that was located between dE6 and dE7, the dE6/7 site (Figures S7A,B).STAT5 binding to this site is consistent with recruitment data for this factor to Ed by ChIP-seq in immune cells and tissues based on ReMap Atlas of Regulatory Regions (Figure S4).Introduction of a mutation that abolished STAT5 binding to the human dE6/7 site (Figure S7C) abrogated enhancer activation by IL-7 treatment in IL7Ra-expressing transfected Jurkat cells (Figure 7B).Together, our results demonstrate that, in addition to the regulation of Tcrg germline transcription and Eg function, IL-7R signaling is crucial for Ed-dependent Tcrd germline transcription. ",
"section_name": "STAT5 binds to Ed",
"section_num": null
},
{
"section_content": "Eg and Ed are regulated in parallel during b-selection, activating germline transcription and VgJg and VdDdJd recombination in DN2b and DN3a thymocytes and gene silencing in DP thymocytes (15,69).Pre-TCR signaling causes dissociation of Eg-and Ed-bound factors in DP thymocytes (14)(15)(16).MYB and RUNX1 dissociate from Eg and Ed during bselection as a result of the pre-TCR-dependent downregulation of Notch1 transcription (14,32), whereas STAT5 dissociates from Eg as a result of terminated Il7ra transcription (16).In this study, we demonstrate that Ed function depends on IL-7Rdependent STAT5 recruitment, similar to the mechanism of Eg function induction, demonstrating a parallel regulatory mechanism of these enhancer functions in controlling Tcrg and Tcrd germline transcription.Hence, the activity of Eg and Ed depends on RUNX1, MYB, and STAT5 recruitment in DN3a thymocytes, whereas these three aforementioned factors dissociate from Eg and Ed in DP thymocytes as a consequence of termination of Notch and IL-7R signaling, revealing the molecular mechanism by which Tcrg and Tcrd transcription is regulated in parallel during thymocyte development. To study the role of the combined effect of IL-7 and Notch signaling, we analyzed the effect of IL-7 on ICN1-transduced SCID.adh cells.These cells produce full-length and truncated Notch1 transcripts, which derive from an intragenic deletion of approximately 38 kb and consist of exon 1 joined to an 81-kb noncontiguous intron 1 sequence that it is spliced to a site 12 bp 3ó f the exon 28 splice acceptor site (70).The resulting polypeptide can insert into the cell membrane due to its hydrophobic Nterminus, driving ICN1 expression to generate ligandindependent signals in a DAPT-sensitive fashion.Previous studies have demonstrated that SCID.adh cells constitutively express some levels of ICN1 and respond to DAPT by downregulating ICN1 expression as well as Notch-dependent genes, such as Cd25, Hes1, Il7ra, Runx1, Tcrd and Tcrg (14,70,71).In addition, these cells respond to IL-7 signaling and have been previously used to analyze its role in regulating Tcrg germline transcription (14,44,45).Therefore, SCID.adh cells constitute an excellent model to study the combined effect of Notch and IL-7 in the regulation of Tcrg and Tcrd germline transcription.Consistent with the induction of Il7ra transcription by Notch, our results revealed that IL-7-dependent activation of Tcrd and Tcrg germline transcription was further activated by ICN1 and inhibited by DAPT in these cells.These results strongly suggest that the Notchdependent effect on Il7ra transcription is responsible for the IL-7dependent Tcrd and Tcrg germline transcription observed upon ICN1 overexpression and DAPT treatment in SCID.adh cells. To demonstrate the essential role for IL-7 signaling in activating Ed and Eg4 in SCID.adh cells, we analyzed H3K4me3 and H3K27ac together with the induction of eRNAs as predictors of enhancer activity (65)(66)(67)(68).In fact, enhancer transcription is considered the best indicator of enhancer activity (65,66).The detection of IL-7-induced Eg and Ed transcripts indicates that this treatment induces an opening in the chromatin structure at the enhancer regions.Although other open regions that could function as cis-regulatory elements are present in the vicinity of Ed and Eg4, as indicated by ATAC-seq and the ENCODE Registry of cCREs, these enhancers concentrate the highest binding density of transcription factors, including the specific binding of p300 and STAT5 (Figures S2,S4,S5).This high density of transcription factors that bind to Ed and Eg4 is consistent with the absence of H3K27ac at the core site of these enhancers, with this histone mark detected in the flanking regions of these enhancers (Figures S4-S6).These analyses confirm that Ed and Eg4 are the main regulatory elements present in the regions analyzed.Because STAT5 specifically binds to Ed and Eg4 and not to other nearby enhancers as analyzed by ChIP-seq based on ReMap Atlas of Regulatory Regions, our data indicating that the measured transcripts are IL-7 responsive strongly support that they constitute true Ed and Eg4 eRNAs.Although the distal enhancer EO581865/enhD, located adjacent to Ed at a distance of approximatelly 100 pb, exhibits some levels of STAT5 binding based on ReMap data, ATAC-seq experiments indicate that the EO581865/endD chromatin is not accessible in DN2b and DN3 thymocytes, indicating that Ed is the relevant enhancer at the Tcrd locus during thymocyte development (Figure S4).The low levels of transcripts detected in Ed and Eg4 surrounding regions by RNA-seq in DN thymocytes are consistent with the expected low abundance of eRNAs (Figure S6).Although the role of eRNAs remains unresolved, they are thought to be relevant to maintaining an open chromatin state that is readily accessible for transcription factors, stabilizing enhancer-promoter looping interactions, promoting the loading of RNA-polymerase 2 to the promoter, and/or releasing a paused promoter to an elongating stage (72)(73)(74).Our experiments do not address the potential roles of these eRNAs on Tcrd and Tcrg transcription, but these transcripts likely contribute to maintaining the opening of enhancer chromatin to facilitate access to transcription factors and cofactors in the activation of their specific promoters. Previous experiments with Il7ra -/-mice demonstrated a strong dependence on IL-7R signaling in the regulation of Tcrg germline transcription and VgJg recombination and little apparent effect on Tcrd recombination (38)(39)(40)(41)(42)48).Although a partial inhibitory effect on VdDdJd might be overlooked in these experiments (38), these results differ from our results, with dramatically reduced Trdd2, Trdj1 and Trdj2 germline transcription observed in Rag2 -/- Il7ra -/-DN3a thymocytes and IL-7R-dependent regulation of Ed.Consistent with the important role played by Ed in promoting chromatin accessibility and activating Trdd2, Trdj1 and Trdj2 germline transcription in a discrete chromatin loop (75), previous experiments with Ed -/-mice demonstrated that this enhancer is important for Tcrd germline transcription and VdDdJd rearrangements in DN3a thymocytes and the generation of gd T lymphocytes (23).Because Tcrd germline transcription primarily depends on Ed function (23), our results demonstrate that IL-7R signaling plays a crucial role in the control of Ed-dependent Tcrd germline transcription in DN3a cells.Therefore, the VdDdJd rearrangements detected in Il7ra -/-mice are most likely the consequence of a very low level of Tcrd germline transcription in Rag2 -/-Il7ra -/-thymocytes; this low level of transcription may open the locus chromatin to permit accessibility of the recombinase machinery.Although profoundly reduced compared to the levels in the control mice, 10-12% thymic and 6-10% splenic gd T lymphocytes were detected in the Ed -/-mice; the presence of some gd T cells in Ed -/-mice suggested the implication of additional elements in activating VdDdJd recombination.Our data indicate that nearly all Tcrd germline transcripts were abrogated in Rag2 -/-Il7ra -/-thymocytes compared to Rag2 -/-thymocytes, including those initiated by the Trdd2 promoter (Figure 4), which had been previously proposed to be a possible candidate for promoting Tcrd germline transcription and VdDdJd recombination in Ed -/-mice (61).The detection of only residual Tcrd transcripts in our analysis of Rag2 -/-Il7ra -/- thymocytes does not support a suggestion of additional IL-7R-independent regulatory elements in the activation of VdDdJd recombination.Since our experiments were focused on the regulation of Tcrd and Tcrg germline transcription that occur prior to VdDdJd and VgJg rearrangements and thus TCRgd expression, these data do not directly address the issue of ab vs. gd T-cell commitment, which is accepted to be regulated by differential signaling strength between TCRgd and pre-TCR expressed on the same T-cell precursors (11)(12)(13). In contrast to its important function in Tcrd germline transcription and VdDdJd recombination in thymocytes, Ed is not required for the transcription of a rearranged Tcrd gene in mature gd T lymphocytes; in fact, Ea is the regulatory element critical for this transcriptional function (23,76).IL-7R signaling does not activate Tcra germline transcription or induce STAT5 binding to Ea in SCID.adh cells (Figures S8A,B).In contrast, IL-7R signaling has been previously shown to be involved in preventing premature VaJa recombination in DN4 thymocytes (28).Therefore, IL-7R signaling is probably not required for rearranged Tcrd transcription in mature gd T cells.Because Ed is critical for the premature VaJa rearrangements that have been detected in Ea -/-mice that result in the detection of Va2 + T lymphocytes in these mice (76)(77)(78), NOTCH1 and IL-7R signaling likely regulate the induction of Ed-dependent VaJa rearrangements in DN3a thymocytes. Comparisons between synthetic and natural enhancers have revealed that enhancer activity is best explained by occupancy of specific binding sites regardless of the binding site position (79).Hence, the combination of multiple transcription factor-binding sites and not their organization underlies the specificity of eukaryotic gene expression regulation (80).In addition, temporal expression of specific transcription factors clearly regulates T-lineage identity and development (24).In this regard, the combination of the essential binding sites for RUNX1, MYB and STAT5 is conserved between Eg and Ed in both mice and humans; however, these sites are positioned differently from STAT5-MYB-RUNX1 sites in Eg and RUNX1-MYB-STAT5 sites in Ed (Figure S7).Therefore, the recruitment of these factors to differently organized binding sites within these enhancers could create efficient regulatory structures that are critical for high Tcrg and Tcrd gene expression in DN2b and DN3a thymocytes and gene silencing in DP thymocytes and ab T lymphocytes.We have not directly addressed the effect of IL-7 on the recruitment of RUNX1 and MYB to Ed in SCID.adh cells; however previous studies have shown that IL-7 treatment does not inhibit the recruitment of RUNX1 and MYB to Eg in these cells (44).The fact that RUNX1, MYB and STAT5 bind to Eg in IL-7-treated SCID.adh cells (44) as well as to Eg and Ed in Rag2 -/-thymocytes (14, 16) supports the hypothesis that these factors are simultaneously recruited to these enhancers in DN3a thymocytes.Furthermore, luciferase assays indicated that STAT5 binding synergistically augmented the activity of Eg activity along with RUNX1 and MYB (44), and IL-7 treatment increased Ed activity (Figure 7), which is absolutely dependent on the presence of intact RUNX1 and MYB binding sites (33)(34)(35)(36).Together, these results strongly suggest that these factors are simultaneously recruited to Ed and Eg to regulate enhancer function in DN3a thymocytes. The functional interconnection of the IL-7R and NOTCH1 signaling pathways is essential for normal T-cell development.When this intersection is defective, lymphopenia can be a result, whereas excessive signaling can lead to the development of T-cell acute lymphoblastic leukemia (81).In fact, constitutive activation of NOTCH1 signaling is the most prominent oncogenic pathway during T-cell transformation in more than 60% of all human T-cell acute lymphoblastic leukemia cases, which are mainly caused by different activating mutations (82).Interestingly, in 70% of the latter, chromosomal translocations are evident during thymocyte development as a result of illegitimate TCR gene recombination, with those involving TCRD predominant in approximately 67% of cases (83,84) and Ed being an important element contributing to genomic instability (85).Our results revealing an important role played by IL-7R signaling in the regulation of Ed-dependent Tcrd germline transcription in DN3a thymocytes might contribute to a better understanding of the causes of this disease. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "We thank Nuno L. Alves (Institute for Molecular and Cell Biology, Porto, Portugal) and RenéA.W. van Lier (Academic Medical Center, Amsterdam, The Netherlands) for the Jurkat-GFP and Jurkat-IL7Ra + GFP clones; JoséZamorano (San Pedro de Alcańtara Hospital, Caćeres, Spain) for his help in STAT5 EMSAs; David L. Wiest (Fox Chase Cancer Center, Philadelphia, PA, USA) for SCID.adhcells; Jonathan C. Aster (Harvard Medical School, Cambridge, MA, USA) for the MigR-GFP and MigR-ICN1-GFP constructs; and Clara Sańchez-Gonzaĺez for animal care. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "This work was funded by grants from the Spanish Ministry of Science and Innovation (BFU2016-79699P and PID2021-128720NB-100), Spanish Scientific Research Council (2019AEP202), and Andalusian Government (P20_01271) to CH-M; the Spanish Ministry of Science and Competitiveness (PID2020-118859GB-100) and Andalusian Government (P20_01269) to CS; and JSPS KANENHI (19K08999) to ST-I.This research was co-funded with European Union funds.AR-C, JL-R, and CH-M are part of CSIC´s Global Health Platform (PTI+ Salud Global) (SGL2103033). ",
"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 animal study was reviewed and approved by Ethical Committee of Consejo Superior de Investigaciones Cientıfcas, Spain Ethical Commitee of Andalusian Government, Spain Animal Care and Use Commitee of Kyoto University, Japan.Written informed consent was obtained from the owners for the participation of their animals in this study. 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. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2022.943510/full#supplementary-material ",
"section_name": "Ethics statement",
"section_num": null
},
{
"section_content": "The animal study was reviewed and approved by Ethical Committee of Consejo Superior de Investigaciones Cientıfcas, Spain Ethical Commitee of Andalusian Government, Spain Animal Care and Use Commitee of Kyoto University, Japan.Written informed consent was obtained from the owners for the participation of their animals in this study. ",
"section_name": "Ethics statement",
"section_num": null
},
{
"section_content": "",
"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
},
{
"section_content": "The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2022.943510/full#supplementary-material ",
"section_name": "Supplementary material",
"section_num": null
}
] |
10.1158/1078-0432.22448781.v1
|
Supplementary Table 2 from CD62L as a Therapeutic Target in Chronic Lymphocytic Leukemia
|
<jats:p><p>Supplementary Table 2 - PDF files 77K, CD markers used in phenotype screen</p></jats:p>
|
[
{
"section_content": "",
"section_name": "",
"section_num": ""
}
] |
[] |
10.3389/fonc.2021.698551
|
Complementary Sequential Circulating Tumor Cell (CTC) and Cell-Free Tumor DNA (ctDNA) Profiling Reveals Metastatic Heterogeneity and Genomic Changes in Lung Cancer and Breast Cancer
|
<jats:sec><jats:title>Introduction</jats:title><jats:p>Circulating tumor cells (CTCs) and cell-free tumor DNA (ctDNA) are tumor components present in circulation. Due to the limited access to both CTC enrichment platforms and ctDNA sequencing in most laboratories, they are rarely analyzed together.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>Concurrent isolation of ctDNA and single CTCs were isolated from lung cancer and breast cancer patients using the combination of size-based and CD45-negative selection method <jats:italic>via</jats:italic> DropCell platform. We performed targeted amplicon sequencing to evaluate the genomic heterogeneity of CTCs and ctDNA in lung cancer and breast cancer patients.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Higher degrees of genomic heterogeneity were observed in CTCs as compared to ctDNA. Several shared alterations present in CTCs and ctDNA were undetected in the primary tumor, highlighting the intra-tumoral heterogeneity of tumor components that were shed into systemic circulation. Accordingly, CTCs and ctDNA displayed higher degree of concordance with the metastatic tumor than the primary tumor. The alterations detected in circulation correlated with worse survival outcome for both lung and breast cancer patients emphasizing the impact of the metastatic phenotype. Notably, evolving genetic signatures were detected in the CTCs and ctDNA samples during the course of treatment and disease progression.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>A standardized sample processing and data analysis workflow for concurrent analysis of CTCs and ctDNA successfully dissected the heterogeneity of metastatic tumor in circulation as well as the progressive genomic changes that may potentially guide the selection of appropriate therapy against evolving tumor clonality.</jats:p></jats:sec>
|
[
{
"section_content": "Introduction: Circulating tumor cells (CTCs) and cell-free tumor DNA (ctDNA) are tumor components present in circulation.Due to the limited access to both CTC enrichment platforms and ctDNA sequencing in most laboratories, they are rarely analyzed together. Methods: Concurrent isolation of ctDNA and single CTCs were isolated from lung cancer and breast cancer patients using the combination of size-based and CD45-negative selection method via DropCell platform.We performed targeted amplicon sequencing to evaluate the genomic heterogeneity of CTCs and ctDNA in lung cancer and breast cancer patients. Results: Higher degrees of genomic heterogeneity were observed in CTCs as compared to ctDNA.Several shared alterations present in CTCs and ctDNA were undetected in the primary tumor, highlighting the intra-tumoral heterogeneity of tumor components that were shed into systemic circulation.Accordingly, CTCs and ctDNA displayed higher degree of concordance with the metastatic tumor than the primary tumor.The alterations detected in circulation correlated with worse survival outcome for both lung and breast cancer patients emphasizing the impact of the metastatic phenotype.Notably, evolving genetic signatures were detected in the CTCs and ctDNA samples during the course of treatment and disease progression. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Tissue biopsies and radiological imaging are routinely utilized by clinicians to monitor treatment efficacy and disease progression in patients.However, tissue biopsies are invasive and often associated with risks and pain or discomfort, while frequent imaging for example with computed tomography may be costly and involves cumulative exposure to ionizing radiation.Furthermore, tissue biopsy may be limited by sampling error as single site biopsy may not reflect the profile of the whole tumor.In contrast, minimally invasive liquid biopsy allows repetitive non-invasive sample collection and offers a broader tumor genomic profile based on shed DNA or cells from the tumor, with potential for better realtime monitoring of treatment efficacy and disease progression. The metastatic spread of cancer is largely due to the shedding of circulating tumor cells (CTCs) from tumors into the blood stream and invasion of distant organs.Characterization of CTCs has provided mutation profiles of emerging tumor subclones that contribute to metastatic spread and resistance to therapy (1,2).The number of CTCs detected in the blood of cancer patients also correlates with inferior treatment response and survival outcomes (3), suggesting that the assessment of CTCs have prognostic and predictive importance in monitoring treatment efficacy (4).However, beyond the quantification of CTCs, molecular analysis of CTCs is essential for improving our understanding of the tumor biology which may in turn have therapeutic implications. Recently, much attention and effort have been focused on utilizing cell free tumor DNA (ctDNA) as liquid biopsy because of its greater accessibility and easier utility.However, caveats remain as ctDNA consists of fragmented DNA from apoptotic and necrotic tumor cells shed into the bloodstream (5), hence it provides limited insight to tumor biology of individual cells.Moreover, genetic signatures obtained from ctDNA are derived from the major clone in the tumor.The signatures of subclonal tumor that do not respond to the treatment and drive disease progression could be missed by ctDNA (6).Hence, additional complementary information from CTCs may be useful. Due to the limited access to both CTC enrichment platforms and ctDNA sequencing in most laboratories, parallel analysis of CTCs and ctDNA is rare.Furthermore, the technology for isolation of rare CTCs at single cell resolution is restricted to costly and complex platforms.In this study, we utilized the single cell isolation platform, DropCell for CTC isolation at single cell resolution.We implemented a standardized sample processing workflow that allowed for concurrent isolation of CTC and ctDNA followed by targeted amplicon sequencing to evaluate the genomic heterogeneity of ctDNA and CTCs in lung and breast cancer patients. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "A total of 16 lung adenocarcinoma and 21 breast ductal carcinoma patients diagnosed at the National Cancer Centre Singapore were recruited.A total of 48 paired tumor tissues were obtained from the surgical resections/biopsies at disease diagnosis and progression.The detailed description of the patient's sample can be obtained in Supplementary Table 1. ",
"section_name": "Patients",
"section_num": null
},
{
"section_content": "A total of 10ml EDTA blood was collected from the patients.We allocated 2.5ml blood for collection of buffy coat as germline DNA control sample, while 7.5ml blood was subjected to CTC enrichment using ClearCell ® FX1 (Biolidics) platform following the manufacturer's recommendation.First, the plasma was isolated by low-speed centrifugation at 500g for 10 minutes.The supernatant was transferred to another 1.5 ml Eppendorf tube followed by high speed centrifugation at 16,000g for 10 minutes at 4°C.The supernatant was snap frozen in dry ice and stored at -80°C.Second, the red blood cells were lysed using RBC lysis buffer (G-Biosciences) in a 1 blood: 3 buffer ratio for 10 minutes.The buffy coat was collected by centrifugation at 500g for 10 minutes.The supernatant was discarded and the buffy coat pellet was resuspended in 4mL of ClearCell FX Resuspension Buffer (Biolidics), followed by CTC enrichment on ClearCell ® FX1 platform using protocol 1. ",
"section_name": "Separation of Plasma and Buffy Coat for Isolation of ctDNA and CTCs",
"section_num": null
},
{
"section_content": "In order to isolate CTCs at single cell resolution, we applied CD45-antibody negative selection using a single cell capture method as described previously (7,8).In brief, larger cells retrieved from the buffy coat were processed through the single cell selection microfluidics.Cells were pre-incubated with CD45-FITC antibodies (Abcam Inc., USA) and positively fluorescent cells were excluded.Cells that fit the criteria of large nucleus to cytoplasmic (N/C) ratio, intact cell membrane and CD45negative fluorescent stains were ejected from the microfluidics platform one at a time for single cell processing. ",
"section_name": "Single Cell Isolation With DropCell",
"section_num": null
},
{
"section_content": "DNA was extracted from the FFPE samples using the GeneRead DNA FFPE Kit (Qiagen).Following the manufacturer's instructions, each section of the FFPE sample underwent deparaffinization and digestion, before allowing the DNA to bind to the QIAamp MinElute column.While bound, the DNA was washed to remove any contaminants.The DNA was then eluted with 25ml of Buffer ATE. ",
"section_name": "DNA Extraction of Formalin-Fixed Paraffin-Embedded (FFPE) Tissues",
"section_num": null
},
{
"section_content": "DNA was extracted from frozen tissue using the AllPrep DNA/ RNA/miRNA Universal kit (Qiagen).Following the manufacturer's instructions, the frozen tissue was first disrupted using the mortar and pestle method, before being homogenized using the QIAshredder (Qiagen).The homogenized lysate was spun in an AllPrep DNA Mini spin column, where DNA was bound to the column and the RNA was found in the flow-through.Total RNA was first purified and bound on the RNeasy mini spin column.While bound, the RNA was washed to remove any contaminants.RNA was eluted in 30ml of RNase-free water.Next, genomic DNA that was bound on the AllPrep DNA Mini spin column was washed to remove any contaminants.50ml of Buffer EB was used to elute the DNA. ",
"section_name": "DNA Extraction of Frozen Tissues",
"section_num": null
},
{
"section_content": "DNA was extracted from plasma using the QIAamp Circulating Nucleic Acid kit (Qiagen).Following the manufacturer's instructions, the sample was first lysed to release DNA bound to proteins, before allowing it to bind to the QIAamp Mini column using the VacConnector on the QIAvac 24 Plus.While bound, the DNA was washed to remove any contaminants.The DNA was eluted with 30ml of Buffer AVE. ",
"section_name": "DNA Extraction of Circulating Cell-Free Nucleic Acid",
"section_num": null
},
{
"section_content": "Whole genome amplification was performed on the isolated single lung CTC samples using REPLI-g single cell kit (Qiagen).The denaturation buffer was added to the DNA followed by a 3 min incubation at room temperature.The denaturation was terminated by addition of neutralization buffer.The DNA amplification was performed in a reaction mix consisting of reaction buffer and DNA polymerase for 1.5 hours at 30°C.The reaction was terminated by inactivation of the DNA polymerase at 65°C for 3 min.The amplified DNA was cleaned using ethanol precipitation. ",
"section_name": "Whole Genome Amplification for Lung CTC Samples",
"section_num": null
},
{
"section_content": "In order to evaluate the quality of the amplified DNA in particularly the possible allelic dropout rate during WGA, we designed a total of 10 primers sets targeting genomics regions of different chromosomes (Supplementary Table 2).The qPCR reactions were carried out using SYBR Green Master Mix (Roche) with 10ng of input DNA and 0.6nM of primers (IDT) on the LightCycler ® 480 platform (Roche) with the following thermocycling conditions: 95°C for 5 mins; 45 cycles of 95°C for 10s and 60°C for 1 min; 95°C for 10s and 65°C for 1 min.Samples with Ct values <35 in at least 5 of the 10 target regions (50% positive rate) were selected for downstream DNA sequencing experiment. ",
"section_name": "Quality Control of Amplified Lung CTC With Quantitative PCR",
"section_num": null
},
{
"section_content": "Whole genome amplification was performed on the isolated single breast CTC samples using Ampli-1 Single Cell WGA kit (Silicon Biosystems).The cell lysis was performed with the addition of Lysis Reaction Mix to each sample.DNA digestion was carried out with the Digestion Reaction Mix followed by ligation with Ligation Reaction Mix.PCR was carried out following the recommended thermo-cycling condition. ",
"section_name": "Whole Genome Amplification for Breast CTC Samples",
"section_num": null
},
{
"section_content": "In order to evaluate the quality of the amplified DNA, we performed PCR experiment on the amplified DNA with Ampli1 QC Kit (Silicon Biosystems) following the manufacturer's recommendation.Samples that gave positive amplification for at least 2 out of the 4 amplicons (50% positive rate) were selected for downstream ampliconsequencing work. ",
"section_name": "Quality Control of Amplified Breast CTC With Quantitative PCR",
"section_num": null
},
{
"section_content": "The EGFR T790M and EGFR exon19 deletion mutation assay were purchased from Life Technologies.ASPCR was conducted on ABI7900 qPCR machine (Life Technologies) using 1x TaqMan Genotyping Master Mix, 1x TaqMan Probe and 20ng of DNA with the following thermal-cycling conditions: 95°C for 10 mins; 5 cycles of 92°C for 15s and 58°C for 1 min; 40 cycles of 92°C for 15s, 60°C for 1 min. ",
"section_name": "Allele Specific PCR (ASPCR)",
"section_num": null
},
{
"section_content": "PCR for detection of EGFR Exon19 deletion mutation was carried out using 0.4µM primers: Forward: 5'-ATGT GGCACCATCTCACAAT-3'; Reverse: 5'-CAGCTGCCAG ACATGAGAAA-3'; 20ng of DNA, 1x AccuTaq LA Buffer (Sigma), 500µM dNTP, 0.05U/µl JumpStart RED AccuTaq LA DNA Polymerase under the following thermo-cycling condition: 94°C for 3 mins; 15 cycles of 94°C for 20s, 58°C for 30s, 68°C for 1 min; 20 cycles of 94°C for 20s, 55°C for 30s, 68°C for 1 min; 68°C for 5 mins.The PCR amplicon was purified with PCR purification kit (Qiagen) following the recommended protocol.The purified PCR product was submitted to Sanger sequencing service provider, Axil Scientific. ",
"section_name": "Sanger Sequencing",
"section_num": null
},
{
"section_content": "We customized 2 different gene panels using Qiagen's GeneRead DNAseq Custom Builder tool.The list of genes and its coverage regions are listed in Supplementary Tables 3,4.Multiplex PCR was performed using GeneRead HotStar Taq DNA polymerase and four primer pools with a total of 80 ng of tumor or CTC DNA and 20ng of cell-free plasma DNA.The amplicons were pooled together and cleaned using AMPure beads (Beckman Coulter).The PCR-enriched DNA was subjected to nextgeneration sequencing library construction using QIAseq 1-Step Amplicon Library Kit (Qiagen).Each library was barcoded with a unique index and quantified using KAPA Library Quantification kit (Kapa Biosystems).Equal amounts of individual libraries were pooled together for a 150bp paired-end sequencing run on the Nextseq (Illumina) platform. ",
"section_name": "GeneRead Targeted DNAseq",
"section_num": null
},
{
"section_content": "The sequenced reads were mapped to the human reference genome hg19 using BWA pipeline (0.7.12) followed by preprocess based on Genome Analysis Toolkit (GATK 3.5) (9) best practices and BAQ (Base Alignment Quality) calculation using SAMtools (1.3) (10).The aligned reads were sorted based on coordinates. ",
"section_name": "Reads Alignment and Base Quality Refinement",
"section_num": null
},
{
"section_content": "We used LoFreq 2.1.1 (11) pipeline for detection of single nucleotide variant (SNV) and insertions/deletions (INDEL) variants with default parameters.We used Quandico 1.13 for copy number variation (CNV) detection with the following modification to the default setting: primer length was set to 21 (average primer length of our GeneRead panel); reads with mapping quality score less than 30 were excluded from the analysis and we grouped the reads into regions as qcluster. ",
"section_name": "Variant Calling and Copy Number Variation Analysis",
"section_num": null
},
{
"section_content": "A reliable analytical pipeline to minimize false positive detection of mutation in our data was developed to address random errors associated with whole genome amplification and sequencing (12), as well as sequencing error/variation due to low abundance of ctDNA samples.Our previous work has conducted a systematic evaluation of the amplification error generated during whole genome amplification using normal DNA as test sample (1).We demonstrated that applying a 10% cut-off on the variant allelic frequency (VAF) of amplified DNA, it could avoid false positive detection of mutation owing to amplification errors.Hence, we applied the same 10% VAF threshold for single CTC that has undergone DNA amplification, and 1% VAF for ctDNA and tumor samples.Further, in order to eliminate false positive detection without missing out genomics alteration due to tumor heterogeneity, we only considered mutations that met the following criteria: shared between tumor and CTC or ctDNA samples; shared between CTC and ctDNA samples; shared between at least two individual CTCs from the same patient (Figure 1F). ",
"section_name": "The Analytical Pipeline for Detection of Mutation and Copy Number Variation",
"section_num": null
},
{
"section_content": "The survival analysis were carried out for top five genes that were altered in lung cancer samples (CSMD2, DMBT1, EGFR, RYR2 and NOTCH1) and breast cancer samples (MTOR, KMT2C, EGFR, ERBB3 and USH2A) using the cBioPortal (13,14) survival analysis tool from a total of 511 lung cancer and 996 breast cancer patients cohort data (15)(16)(17)(18)(19)(20)(21). ",
"section_name": "Survival Analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "We obtained single time point clinical specimens from 16 lung cancer and 5 breast cancer patients from a single comprehensive cancer centre.In addition, serially collected tumors and blood samples were collected from another 16 breast cancer patients.The clinicopathological information of these patients are described in Tables 1,2.The baseline specimens were collected at baseline prior to initiation of specific line of systemic anticancer therapy.Additional blood samples were collected after two cycles while on therapy where feasible, as well as at first response assessment and eventually at disease progression or completion of therapy. CTCs display dynamic phenotypic changes from epithelial to mesenchymal and vice versa during the different stages of disease progression (22).Here, we utilized a combination of size-based and CD45-negative selection method to enrich for CTCs In order to ensure sample comparison validity, CTCs and ctDNA were isolated from the same blood tube.The buffy coat component was used to isolate single CTCs using ClearCell FX and DropCell platforms while the plasma portion was used in ctDNA detection (Figures 1A,B).A total of 116 CTCs and 41 plasma were collected from lung cancer patients while 159 CTCs and 53 plasma were obtained from breast cancer patients (Supplementary Table 5).Upon implementing the analytical pipeline to eliminate the whole genome amplification and sequencing errors, we kept a total of 16 tumors, 39 CTCs and 14 ctDNA from 16 lung cancer patients; a total of 32 tumors, 108 CTCs and 35 ctDNA from 21 breast cancer patients in the final data set. We detected the presence of EGFR T790M mutation in the isolated ctDNA (Figure 1C) as well as the EGFR exon 19 deletion using allelic-specific PCR (ASPCR) and Sanger sequencing on single cells isolated from a lung cancer patient with known EGFR exon 19 deletion mutation (Figures 1D,E) using this workflow, proving the capability of the technology for this purpose that is in concordant with previously reported studies from other cohorts of lung and breast cancer patients (7,23). ",
"section_name": "Establishing a Systematic Workflow for Isolation of ctDNA and Single CTCs",
"section_num": null
},
{
"section_content": "In order to assess the mutation profiles of CTCs, ctDNAs and tumors, we custom-designed targeted gene panels for ampliconsequencing.The lung cancer GeneRead panel consisted of the 45 most frequently mutated or druggable genes in lung cancer as previously reported (24,25) with a targeted region of ~207kb.The breast cancer GeneRead panel consisted of the 58 most frequently mutated or druggable genes in breast cancer as reported by TCGA (26) with a targeted region of ~233kb.We found that single nucleotide variation (SNV) or insertion/ deletion (INDEL) mutations were frequently detected in EGFR, CSMD2, BRAF, TP53 and RYR2 genes in lung cancer samples (Figure 2 and Supplementary Table 6), while frequent copy number alterations were found in RYR2, RELN, DMBT1, CSMD2 and NOTCH1 (Supplementary Table 7).In contrast, we noted that the SNV or INDEL mutations were commonly detected in MYC, TP53, USH2A, NOTCH1 and PIK3CA genes in breast cancer samples (Supplementary Table 8), while MTOR, KMT2C, EGFR, USH2A and NF1 amplification were frequently found in breast cancer samples (Figure 3 and Supplementary Table 9). ",
"section_name": "The Molecular Profiling of the Tumours, CTCs and ctDNA",
"section_num": null
},
{
"section_content": "Though intratumoral heterogeneity (ITH) has been well studied in lung and breast cancers (25,(27)(28)(29), the comparative degree of heterogeneity between the CTCs, ctDNA and tumor remains poorly described.The distribution of the genomic alterations present in all the corresponding tumors, CTCs and ctDNAs collected for each patient are shown in Figure 4. We observed a high degree of heterogeneity in the mutation profiles of CTCs that were undetected in the matched tumor.We found that 78% of the lung CTCs and 91% of the breast CTCs had at least one shared mutation with the matched tumors (Tables 3,4).In contrast, we observed that all (100%) of the lung and breast ctDNA have at least one shared genomic alteration with the matched tumor (either primary tumor and/or metastatic lesion).Moreover, we noted that some mutations shared between CTCs and ctDNA were absent in the tumor.These findings highlight the heterogeneity of tumor subclones shed into circulation. Of note, two lung cancer patients (L6 and L13) had more than 50% of the detected mutations found privately only in the CTCs and not in ctDNA.Both of these patients had a survival shorter FIGURE 1 | The workflow for sample's isolation, CTC mutation validation and analysis pipeline used in this study.(A) The isolation of CTCs and ctDNA from the same tube of blood.CTC was enriched through the ClearCell FX system.(B) CTC was isolated on single cell resolution using the DropCell platform.An image of the cells captured in the cell chamber.(C) The detection of EGFR T790M mutation with ASPCR in the negative control (GM12878 cell); positive control (H1975 cell) and ctDNA from a lung cancer patient.(D) The presence of EGFR Exon19 deletion mutation with ASPCR in the negative control (GM12878 cell); positive control (PC9 cell) and two CTCs isolated with the DropCell platform from a lung cancer patient.(E) The electropherogam of Sanger sequencing run for validation of EGFR Exon19 deletion mutation in negative control (GM12878 cell); positive control (PC9 cell) and two CTCs from a lung cancer patient with known EGFR Exon19 deletion mutation.(F) The analysis pipeline used in this study.than one year, indicating that the highly heterogeneous profiles of CTCs may provide information to stratify patients with poorer prognosis.We also noted more private mutations in the CTCs and ctDNAs for breast cancer samples, at least partly due to increased number of CTCs and ctDNA analyzed in breast cancer as compared to lung cancer samples.We did not detect any clear trend for the degree of heterogeneity with patient survival outcome for breast cancer samples.Longer follow up and larger sample sizes will be required to investigate the association with survival outcome. ",
"section_name": "The Heterogeneity of Genomic Alterations in Tumour, CTCs and ctDNA",
"section_num": null
},
{
"section_content": "The mutation profiles of CTCs, ctDNA, matched primary and metastatic tumors of selected breast cancer patients were further compared spatially and temporally.Where the degree of mutational heterogeneity between paired primary and metastatic tumors is low, mutation profiles found in the CTCs and ctDNA were similar to the tumors (Figure 5A).In contrast, in primary and metastatic tumors that exhibited a high degree of mutational heterogeneity (Figure 5B), CTCs and ctDNA displayed higher degree of resemblance to metastatic tumor than the primary tumor, indicating that CTCs and ctDNA are potentially valuable resources to inform on the heterogeneity of metastatic tumor. In order to investigate the temporal genetic signatures during the course of treatment and disease progression stages, we obtained serial collection of tumor, CTCs and ctDNA of different treatment cycles from a subset of breast cancer patients (n=15).The longitudinal follow up of CTC count and ctDNA CNV displayed differential correlation with the CA15.3 biomarker (Figure 5C).We found that the majority of DNA aberrations were consistently detected at different time points (Figure 5D).Notably, we observed several CNV such as JAK3, BRAF or MTOR amplifications were present only at selected time points of the collected CTCs but not found in the matched ctDNA.We posit that this observation could be explained by the viable CTCs that have evolved and survived the treatment while ctDNA provided genetic information of apoptotic tumor cells.Further, we found emerging of new JAK2 and ATM alterations in patient B8 at the second time point (Figure 5E), indicating possible evolving tumor clonal growth during disease progression. ",
"section_name": "CTCs and ctDNA Provide Complementary Genomic Information of Metastatic Tumour and Evolving Genetic Signatures During Disease Progression",
"section_num": null
},
{
"section_content": "We further explored whether the concomitant genomic alterations that present at high frequency in the tumor, CTCs and ctDNA, may provide information that is linked to patient survival outcome.Taking the top 5 most frequently concomitant altered genes detected in lung cancer CTC/ctDNA samples (CSMD2, DMBT1, EGFR, RYR2 and NOTCH1) and breast cancer CTC/ctDNA samples (MTOR, KMT2C, EGFR, ERBB3 and USH2A) from our study, we applied this gene set to a curated database of lung (n=511) or breast (n=996) cancers (13)(14)(15)(16)(17)(18)(19)(20)(21) obtained from cBioPortal (13,14) for survival analysis.We found that these gene sets were significantly associated with worse survival outcome (Figures 6A,B).In addition, we found significant increase of copy number alterations in the CTC and tumor samples when the disease progressed (Figures 6C-H). The absence of such observation in ctDNA could be explained by its smaller sample size.Cumulatively, this indicates that the mutation profiling of CTCs and ctDNA is useful to provide genetic information with prognostic relevance. ",
"section_name": "The Mutation Profiles of CTC, ctDNA and Tumour Displayed Signatures Associated With Patient Survival Outcome and Disease Progression",
"section_num": null
},
{
"section_content": "CTCs and ctDNA represent different tumor components present in the circulation of cancer patients.Relatively few studies have analyzed both CTCs and ctDNA concurrently, hence their similarity or differences remains largely unknown.Though a few previous publications have analyzed CTCs, ctDNA and tumor from the same patient, these studies focused on new (32) taken from the same source sample, the similar approach on other solid tumors is lacking.To address this, we obtained CTCs and ctDNA from the same source sample, and used label-free CTC isolation followed by CD45-negative selection of CTCs at single cell resolution with a new single cell isolation platform, DropCell, to provide agnostic selection regardless of epithelial or mesenchymal phenotype.We had recently described the heterogeneity in gene expression of single CTCs relative to intra-tumoral heterogeneity, between metastatic and non-metastatic lung cancer (33), as well as comparing the drug-responsive and drug-resistant lung cancer lines (34).Here we found that the majority of the CTCs and ctDNA displayed similar mutation profiles with the matched tumor.However, we noted some private mutations that were found only in two or more CTCs from the same patients, consistent with other studies (30,32).This highlights that CTC heterogeneity could be dependent on tumor heterogeneity and clonal selection.In contrast, we observed less heterogeneity in the ctDNA as compared to CTCs, indicating possibly that the ctDNA was derived largely from the dominant clone in the tumor.Intriguingly, patients with distant metastasis to multiple organs displayed a higher degree of heterogeneity in tumor and CTCs, suggesting that subclonal evolution in tumor progression also featured in CTCs.This was further supported by an evolving genomic signature in sequential samples of CTCs and ctDNA.Hence, the ability to characterize the cells with metastatic properties could provide useful insights for better management of cancer in patients.Future studies would be required to define the metastatic heterogeneity of the CTCs and the association with poorer survival in tumors exhibiting the heterogeneity observed here. Finally, frequent genetic alterations detected in lung and breast cancer have been reported by previous studies to be associated with poor prognosis in lung or breast cancer patients (35)(36)(37)(38)(39)(40)(41)(42)(43).Accordingly, we found that the top five most frequently mutated genes in both CTCs and ctDNA had prognostic value when applied to existing cancer cohorts.Indeed, the alterations found in these genes such as RYR2, NOTCH1 and DMBT1 have strong association with high tumor burden and tumorigenesis (44)(45)(46)(47), possibly resulting in a worse prognosis in lung cancer patients.Further, frequent alterations of USH2A, EGFR and ERBB3 were previously reported in association with cellular proliferation (48)(49)(50)(51).Further, we observed significant increase of copy number changes in the tumor and CTCs during disease progression.This finding suggests that incorporating the mutational profile for the CTCs in addition to enumeration, could build upon existing prognostic variables defined by FDA approved systems such as CellSearch, and potentially better defining the groups that need to receive adjuvant therapy. Besides molecular characterization, the functional analysis of the identified mutations could provide additional insights to assess the consequences of these mutations in cancer therapy.We have previously developed CORTAD-seq that allows for concurrent analysis of transcriptome and mutation at single cell resolution (34).Using this tool, we found that single cell that harbors selected tyrosine kinase inhibitor (TKI)-responsive or -resistance EGFR mutations possesses differential transcriptomic signatures upon TKI stimulation.Hence, we envision that future study incorporating CORTAD-seq or other similar tool will be useful to dissect the consequences of mutation on cellular phenotypes.We acknowledge that a technical limitation in our study is the low number of CTCs that passed the quality control check.Only ~34% of all the isolated lung CTCs and ~68% of all the isolated breast CTCs were kept in the final analysis (Supplementary Table 6).This observation is in concordant with the findings by Dirix's and Park's laboratories where 43 to 60% of the isolated CTCs yielded positive result from the downstream transcriptional and mutational analysis (52,53), highlighting that it is important to improve the quality of the amplified DNA from the CTCs sample.We believe that this is a technological limitation that can be overcome with improving single cell analytical platforms that could be mated with this workflow.Cumulatively, we present a workflow for robust simultaneous evaluation of CTCs and ctDNA from the same source sample.The molecular profiling of serially collected liquid biopsies could inform and build on existing algorithms for prognostication and management using CTCs and ctDNA.The remaining 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. Copyright © 2021 Kong, Liu, Tan, Tai, Phua, Poh, Yeo, Chua, Haw, Ling, Ng, Tan, Loh, Tan, Ng, Ang, Toh, Lee, Lim, Lim, Hillmer, Yap and Lim.This is an open-access 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": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "We would like to thank Ms Wang Lan Ying, Thum Chooi Mei, Maria Rosita Ibarola Negapatan, Zhou Qi, Fong Yian Ching and Mr Eric Wen Jian Soong for assistance in coordinating patient's consent and sample's collection. ",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
},
{
"section_content": "FUNDING This work is supported by the Career Development Award (14302FG096) by Joint Council Office (JCO), Agency for Science, Technology and Research, Singapore to SK; Young Individual Research Grant (NMRC/OFYIRG/0056/2017) by National Medical Research Council (NMRC), Singapore for SK; Clinician Scientist Award (MOHIAFCat1/0034/2015 and CSA-INV/0025/2017) by NMRC, Singapore for W-TL; Clinician Scientist Award (MOH-CSAINV18nov-0009) for YY by NMRC; the infrastructure of Lung Cancer Consortium Singapore that is jointly funded by Open Fund Large Collaboration Grant (NMRC/OFLCG/002/2018) by NMRC, Singapore; Trailblazer Foundation, Singapore Millennium Foundation, and the National Cancer Centre Research Fund. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The original contributions presented in the study are included in the article/Supplementary Material.Further inquiries can be directed to the corresponding authors. ",
"section_name": "DATA AVAILABILITY STATEMENT",
"section_num": null
},
{
"section_content": "All patients gave written informed consent to participate in this study and the biological samples were collected from the patients following the Institutional Review Board (IRB) approval.The patients/participants provided their written informed consent to participate in this study. SK, JT, LP, HP, and YH performed the experiments described in this study.XL, SK, LP, and WL performed the data analysis.ST, TY, and YC processed the CTC enrichment and single cell isolation experiment with the technical support from YL. RN, KL, MA, QN, DT, W-TL, and YY recruited patients for this study.CL, TL, and AH provided guidance and support to this work.SK wrote the manuscript with the inputs from XL, ST, W-TL, and YY.All authors contributed to the article and approved the submitted version. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2021.698551/full#supplementary-material ",
"section_name": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "All patients gave written informed consent to participate in this study and the biological samples were collected from the patients following the Institutional Review Board (IRB) approval.The patients/participants provided their written informed consent to participate in this study. ",
"section_name": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "SK, JT, LP, HP, and YH performed the experiments described in this study.XL, SK, LP, and WL performed the data analysis.ST, TY, and YC processed the CTC enrichment and single cell isolation experiment with the technical support from YL. RN, KL, MA, QN, DT, W-TL, and YY recruited patients for this study.CL, TL, and AH provided guidance and support to this work.SK wrote the manuscript with the inputs from XL, ST, W-TL, and YY.All authors contributed to the article and approved the submitted version. ",
"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/fonc.2021.698551/full#supplementary-material ",
"section_name": "SUPPLEMENTARY MATERIAL",
"section_num": null
}
] |
10.3389/fonc.2019.01268
|
Cationic Amino Acid Transporter-1-Mediated Arginine Uptake Is Essential for Chronic Lymphocytic Leukemia Cell Proliferation and Viability
|
Interfering with tumor metabolism by specifically restricting the availability of extracellular nutrients is a rapidly emerging field of cancer research. A variety of tumor entities depend on the uptake of the amino acid arginine since they have lost the ability to synthesize it endogenously, that is they do not express the rate limiting enzyme for arginine synthesis, argininosuccinate synthase (ASS). Arginine transport through the plasma membrane of mammalian cells is mediated by eight different transporters that belong to two solute carrier (SLC) families. In the present study we found that the proliferation of primary as well as immortalized chronic lymphocytic leukemia (CLL) cells depends on the availability of extracellular arginine and that primary CLL cells do not express ASS and are therefore arginine-auxotrophic. The cationic amino acid transporter-1 (CAT-1) was the only arginine importer expressed in CLL cells. Lentiviral-mediated downregulation of the CAT-1 transporter in HG3 CLL cells significantly reduced arginine uptake, abolished cell proliferation and impaired cell viability. In a murine CLL xenograft model, tumor growth was significantly suppressed upon induced downregulation of CAT-1 in the CLL cells. Our results suggest that inhibition of CAT-1 is a promising new therapeutic approach for CLL.
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western World.It is characterized by the accumulation of mature, typically CD5 + CD23 + neoplastic B-lymphocytes in the peripheral blood, lymph nodes, bone marrow and/or other lymphoid organs (1).Despite tremendous progress in treatment options by B cell receptor signal transduction inhibitors (ibrutinib, idelalisib) and bcl2 inhibitors (venetoclax), it still remains an incurable disease that needs novel treatment approaches. One of the key hallmarks of a cancer cell is its metabolic reprogramming that supports proliferation and acquisition of biomass (2).This offers the possibility to interfere therapeutically with non-redundantly up-or downregulated branches of the rewired cancer cell metabolism (3,4).The increased demand of cancer cells for nutrients is apparent in the well-established enhanced uptake of glucose in conjunction with its preferential use via aerobic glycolysis, referred to as the Warburg effect (5).Amino acids are another important class of nutrients, which cancer cells need to acquire and metabolize for viability and continuous growth (5,6).Asparagine depletion via asparaginase in acute lymphoblastic leukemia (ALL) is an example for interference with amino acid availability as clinically established anti-tumor strategy.Cysteine depletion by cysteinase is in preclinical development for CLL (7) based on the inherent dependency of CLL cells on cysteine for glutathione synthesis (8). Arginine, a semi-essential proteinogenic amino acid that becomes limiting only in situations of increased demand, is a substrate for metabolic pathways leading to e.g., nitric oxide (NO), polyamines and collagen (9,10), and is also a key activator of mammalian target of rapamycin (mTOR), a crucial metabolic checkpoint of cellular proliferation (11).The availability of arginine is essential for the proliferation and function of physiological immune cells (12,13) and is also a decisive regulatory parameter within the tumor microenvironment: both, tumor cells and immune cells compete for this amino acid (13)(14)(15)(16).Most tissues constitutively or inducibly express the enzymes argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL), which metabolize the non-proteinogenic amino acid citrulline to arginine in a two-step reaction (17,18).In contrast, various tumor entities do not express ASS and therefore depend on the availability and uptake of extracellular arginine.This socalled arginine-auxotrophy (19) has been shown for solid cancer entities (e.g., melanoma, sarcoma, hepatocellular carcinoma) (20), but has more recently also been described for hematological cancers like acute myeloid leukemia (21) or acute lymphoblastic leukemia (22).Restricting the availability of arginine by the systemic administration of the arginine-metabolizing enzymes arginine deiminase (ADI) (23,24) or arginase (25) is already in clinical development with promising results. A novel therapeutic strategy to deprive cancer cells of arginine would be to interfere with its uptake (5) if (i) a defined arginine transporter would be overexpressed in a tumor entity compared to physiological tissues and more importantly, (ii) restriction of the availability of arginine upon blockade of this transporter could be compensated by physiological tissues as opposed to cancer cells (26).Arginine transport through the plasma membrane of mammalian cells is mediated by eight different transporters, with different tissue distribution (27,28).The cationic amino acid transporters (CAT-1, CAT-2A, CAT-2B, and CAT-3; SLC7A1-3) recognize exclusively cationic amino acids as their substrates, while the heteromeric amino acid transporters y + LAT1 (SLC7A7), y + LAT2 (SLC7A6), and b 0,+ AT (SLC7A9), as well as ATB 0,+ (SLC6A14) also transport neutral amino acids (27,29).CATs can mediate unidirectional arginine transport into cells (27, 28) and are the main uptake transporters in most mammalian cells.In contrast, y + LATs are obligatory exchangers. They catalyze primarily arginine export in exchange with neutral amino acids and sodium under physiological conditions (27, 28). Studies on amino acid transporters in cancer have so far mainly focused on glutamine transport mediated by ATB 0,+ (30) as well as the exclusive neutral amino acid transporters ASCT2 (SLC1A5) (31, 32) and LAT1 (SLC7A5) (33) showing a dependence of cancer cell growth on transporter-mediated amino acid uptake.However, the expression, regulation and potential non-redundant function of defined arginine transporters for cancer cell viability and growth has not been studied so far in hematological cancer entities.Here we analyzed the expression of arginine transporters in human primary CLL cells as well as CLL cell lines.We found a non-redundant role of the arginine transporter CAT-1 for CLL cell viability and proliferation.Our data address previously unrecognized aspects of CLL cell physiology and-more importantly-identify a potential novel therapeutic target structure for CLL therapy. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "Unless mentioned otherwise, reagents were purchased from Roth (Karlsruhe, Germany) or Sigma Aldrich (Steinheim, Germany).Amino acids were L-isomers. ",
"section_name": "Reagents",
"section_num": null
},
{
"section_content": "This study was approved by Rhineland-Palatinate Medical Association Ethics Committee.Blood donors gave written informed consent in accordance with the Declaration of Helsinki.Primary CLL cells were isolated from peripheral blood of CLL patients (Supplementary Table S1) by Ficoll density gradient centrifugation and harvested from the interphase (fraction of CD5 + CD19 + cells > 95% within the mononuclear cell population in all cases).Activation of CLL cells (6 × 10 6 /ml) was induced by 7.5 µg/ml Toll-like receptor (TLR)9 agonist ODN2006 (InvivoGen, San Diego, CA) (34).CLL cell lines HG3, MEC1, JVM-2, and primary CLL cells were cultured in RPMI 1640, supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin.For arginine starvation, cells were incubated as described (18). ",
"section_name": "Primary Cells and Cell Lines",
"section_num": null
},
{
"section_content": "Cell proliferation was assessed by the incorporation of [ 3 H]thymidine as described (18).Cell viability was determined using the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, CA). ",
"section_name": "Proliferation and Cell Viability Assays",
"section_num": null
},
{
"section_content": "Uptake of 100 µM [ 3 H]arginine (10 µCi/ml, Santa Ana, CA) was measured as described for citrulline uptake in T cells (18), except that 0.5 × 10 6 HG3 cells or 1 × 10 6 primary CLL cells were analyzed per well. ",
"section_name": "Arginine Uptake",
"section_num": null
},
{
"section_content": "Immunoblotting was performed as described (18,35) using CAT-1 (1:5,000), y + LAT1 (1:3000) or y + LAT2 (1:10,000) antibodies produced in the laboratory of Ellen Closs, as well as ASS (Sigma Aldrich, HPA020896, 1:1,000) or GAPDH antibodies (Cell Signaling, Danvers, MA, 14C10, 1:10,000). ",
"section_name": "Immunoblotting",
"section_num": null
},
{
"section_content": "Transporter mRNA expression was analyzed by two-step qRT-PCR (36).Primers and hybridization probes were previously described (35, 37) except for y + LAT2 (ss: 5 ′ CACGTTCACTTA CGCCAAGGT, as: 5 ′ TCAGAGTGTCCCTGGCACAGT, probe: 5 ′ TGCCATCATTGTCATGGGCCTTGTTA). Transporter mRNA copies were quantified using in vitro-synthesized RNAs, containing the complete coding region of each transporter (37). ",
"section_name": "Quantitative Real Time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)",
"section_num": null
},
{
"section_content": "Virus production and transduction were performed as described (38).Virus particles, carrying CAT-1 shRNA for constitutive expression were produced from HEK293T cells, cotransfected with pCMV R8.9, pCMV-VSV-G and pLKO.1-puro_SLC7A1-5(shRNA: TRCN0000042967) (39) or pLKO.1-puro_SHC002(non-target shRNA, Sigma Aldrich). 1 × 10 6 HG3 cells (0.5 ml) were transduced with 2 ml virus supernatant.5 h later, 1 ml of supernatant was replaced by fresh medium.One day later, transduced cells were selected by 5 µg/ml puromycin in the presence of 1 mM citrulline and 67 µM lysine tripeptide for 3 days before starting further experiments.For inducible CAT-1 knock-down, the same shRNA sequence was inserted in Tet-pLKO-puro vector (Addgene plasmid #21915) (40).Virus production was performed with HEK293T cells, cotransfected with psPAX2, pMD2.G, and Tet-pLKO-puro-sh_hCAT1 or Tet-pLKO-puro-scr as control.Transduced cells were selected by 5 µg/ml puromycin for 2 weeks before single cell clones were expanded and characterized.CAT-1 knockdown was induced by 1 µg/ml doxycycline. ",
"section_name": "Lentiviral Transduction of HG3 Cells",
"section_num": null
},
{
"section_content": "NOD.Cg-Prkdc SCID Il2rg tm1Wjl /SzJ (NSG) mice were housed under conditions according to the guidelines for animal care of the animal facility of the Johannes Gutenberg University.Experimental procedures were performed according to German regulations for the use of laboratory animals.2.5 × 10 6 Tet-pLKO-puro-sh_hCAT1 or Tet-pLKO-puro-scr cells were injected subcutaneously in the left flank of NSG mice.ShRNA expression was induced by 1 mg/ml doxycycline and 2% sucrose in drinking water. ",
"section_name": "Mice",
"section_num": null
},
{
"section_content": "Statistical analyses were performed with the GraphPad Prism software 6. Results are expressed as means ± SD.Statistical differences were calculated using one-way analysis of variance (ANOVA), followed by the Tukey post-hoc test, or using ttest, as appropriate.Statistical comparison of survival curves was performed with Log-rank (Mantel-Cox) test.The levels of significance were specified as * * * p < 0.001, * * p < 0.01, and * p < 0.05. ",
"section_name": "Statistical Analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "We first studied the influence of arginine availability on primary human CLL cells, isolated from the peripheral blood (PB) of highly leukemic CLL patients.In CLL, the proliferating fraction is in the bone marrow and in the lymph nodes, while the cells in the blood are arrested in G 0 /G 1 phase (34, 41), PB-derived CLL cells do therefore not proliferate in vitro but can be activated by surface Ig-crosslinking or by triggering TLR9 (34).Upon TLR9mediated CLL cell activation in conventional cell culture medium containing 1 mM arginine, CLL cells entered the cell cycle and proliferation could be detected (Figure 1A).In the absence of arginine, this proliferative response was completely abolished (Figure 1A).CLL viability was not modulated by the absence of arginine within 48 h (Figure 1B). Since ASS expression and functional arginine auxotrophy have not been studied in CLL so far, we analyzed this metabolic feature in primary PB-derived CLL cells.In CLL samples of 18 consecutive patients (Supplementary Table S1), we only saw ASS protein expression in one sample (patient 14; Figure 1C).Upon arginine depletion, tumor cells sometimes induce or upregulate ASS (20).We therefore analyzed if such a metabolic rescue strategy occurs in CLL cells.When primary CLL cells were TLR9-activated for 48 h, ASS was not induced, even under arginine depletion (Figure 1D). Next, we analyzed arginine dependence and ASS expression in human HG3 CLL cells.Arginine depletion for 48 h led to a nearly complete inhibition of HG3 cell proliferation (Figure 2A) consistent with our observation in primary CLL cells (Figure 1A).In parallel, there was a significant induction of cell death as measured by Annexin V (Figure 2B) and propidium iodide (PI) staining (Figure 2C).Comparable results were seen with the CLL cell lines MEC1 and JVM-2 (Supplementary Figures S1A-F).In contrast to the primary activated CLL cells (Figure 1D), in HG3 cells a moderate, time-dependent induction of ASS was noted upon arginine depletion, both, in the presence and absence of citrulline (Figures 2D,E).These results were recapitulated in MEC1 cells (Supplementary Figures S1G,H).Corresponding to the induction of ASS, a high supraphysiological concentration of supplemented citrulline (500-1,000 µM) induced full rescue of HG3 cell proliferation in the complete absence of arginine (Figure 2F).In contrast, at lower supplemented citrulline concentrations (20-40 µM), no relevant augmentation of HG3 cell proliferation was achieved (Figure 2F).Since PB citrulline concentrations in humans are in the range of 20 µM (42), we hypothesize that even under conditions of moderate ASS expression in tumor cells, these cells can still be considered arginine-auxotrophic with full dependence on arginine import. ",
"section_name": "Arginine Availability Is Necessary for CLL Cell Proliferation",
"section_num": null
},
{
"section_content": "After establishing the necessity of extracellular arginine availability for CLL cell proliferation, we analyzed the mRNA expression levels of all potential arginine transporters in unstimulated primary CLL cells.We saw mRNA expression of CAT-1, y + LAT1, and y + LAT2 in all 18 consecutive CLL samples from individual patients (Figures 3A-C).No mRNA expression of CAT-2A, CAT-2B, CAT-3, b 0,+ AT, or ATB 0,+ was detectable in any patient-derived CLL cell sample (data not shown).Protein expression of CAT-1, y + LAT1, and y + LAT2 was seen in all CLL samples, though at varying amounts and with clear interindividual differences.Also, the ratio of the expression of the individual arginine transporters to each other varied between patients (Figure 3D).In these primary CLL cells we saw a time-dependent uptake of arginine (Figure 3E).Upon TLR9-mediated activation an induction of CAT-1 mRNA was detected in all 4 tested individual CLL samples, while y + LAT1 mRNA was decreased and y + LAT2 mRNA was not affected (Figures 3F-H).Also, no mRNA expression of CAT-2A, CAT-2B, CAT-3, b 0,+ AT, or ATB 0,+ was detectable upon stimulation (data not shown).The very prominent activationinduced expression of CAT-1 was also seen on the protein level, while y + LAT1 and y + LAT2 protein expression was only slightly decreased and increased, respectively (Figure 3I).CAT activity, identified as arginine transport insensitive to the competitive inhibition by the neutral amino acid leucine, was 6.5 ± 2.7fold enhanced upon TLR9-mediated activation (Figure 3J).This correlated with the activation-mediated induction of CAT-1 (Figures 3F,I) and the proliferative response of the primary CLL cells (Figure 1A).The induced arginine transport was strongly inhibited by N-ethylmaleimide (NEM) that blocks CAT-, but not y + LAT-mediated transport (Figure 3J) (43). We next analyzed the mRNA expression levels of all potential arginine transporters in HG3, MEC1, and JVM-2 CLL cells and detected a comparable expression pattern to the primary cells (Figure 4A): among the CAT subfamily of arginine transporters, only CAT-1 was expressed.The arginine exporters y + LAT1 and y + LAT2 were also expressed, while no expression was detectable for b 0,+ AT and ATB 0,+ .The mRNA expression pattern was mirrored on the protein level (Figure 4B).In summary, the arginine transporter expression profile of the immortalized cell lines nicely reflected the profile in the primary human CLL cells.Arginine import into HG3 cells (Figure 4C) was about 14 times higher compared to resting human primary CLL cells (Figure 3E) (159.8 ± 86.07 vs. 11.14 ± 6.77 pmol/10 6 cells at 30 s).Arginine uptake was independent of sodium (Figure 4D), excluding the arginine transporter system ATB 0,+ .The CAT inhibitor NEM (200 µM) reduced arginine import significantly by 75.8 ± 4.4% (Figure 4E).The addition of 1 mM leucine to NEM led to an almost complete inhibition of the arginine transport (92.7 ± 2.1%; Figure 4F).Arginine import in HG3 CLL cells was thus mainly mediated via a CAT-based transport activity, in line with our CAT1 mRNA and protein expression data (Figures 4A,B).As y + LATs were the only other arginine transporters expressed in HG3 cells, the minor fraction inhibited by the neutral amino acid leucine was most likely due to y + LAT activity.This y + LAT-mediated arginine uptake is only seen in the experimental setting with no competing neutral amino acids, while these transporters function mainly as arginine exporters under physiological conditions with surplus neutral amino acids in the extracellular compartment. ",
"section_name": "CAT-1 Is the Unique Arginine Importer in CLL Cells",
"section_num": null
},
{
"section_content": "After delineating CAT-1 as the sole transmembrane arginine importer in resting and activated human CLL cells, we addressed its causative role for CLL proliferation and viability.Out of five different plasmids encoding shRNAs to downregulate CAT-1 upon lentiviral transduction, plasmid pLKO.1-puro_SLC7A1-5maximally suppressed CAT-1 mRNA in HG3 CLL cells and was used for all further experiments.Because of the strong inhibition of HG3 cell proliferation in the absence of arginine, we anticipated an inherent inability to expand HG3 clones successfully transduced with CAT-1 shRNA, thereby lacking the potential non-redundant arginine importer.To circumvent this fundamental problem, we expanded the SLC7A1-5-transduced HG3 cells (CAT-1 k.o.) in cell culture medium supplemented with supraphysiological amounts of citrulline (1 mM), since HG3 cells upregulated ASS under intracellular arginine limitation (which should be present in the absence of CAT-1) (Figure 2E) and were able to proliferate even in the complete absence of arginine when 1 mM citrulline was substituted (Figure 2F). Since CAT-1 is also an import transporter for the amino acid lysine, we further added lysine-tripeptide to enable uptake of this amino acid via a peptide transporter (independent of CAT-1 expression). The successfully expanded HG3 CAT-1 k.o. cells exhibited a suppression of CAT-1 mRNA by 80.3 ± 7.9% (Figure 5A).To study potential compensatory regulation, we analyzed mRNA expression of all arginine transporters.While the y + LAT1 expression level was unaffected and y + LAT2 mRNA was increased by 124.6 ± 76.0%, no other arginine transporter was induced as response toward CAT-1 suppression (Figure 5A).On the protein level, we realized a severe loss of the CAT-1 transporter (Figures 5B,C; 90.3 ± 3.2% decrease) upon shRNAmediated knockdown.In contrast to the mRNA data, a slight reduction of y + LAT1 protein expression (28.2 ± 21.6% decrease) was detected, while y + LAT2 protein expression remained unaltered (Figures 5B,C).Since y + LAT transporters function as arginine exporters, this slight downregulation of y + LAT1 can be interpreted as a potential compensatory mechanism to preserve intracellular arginine levels in the context of severely reduced import, resulting from the dramatic reduction of CAT-1 transporter expression of the cells.Upon CAT-1 knockdown, HG3 cells indeed demonstrated significantly reduced system y + activity, determined as leucine-insensitive arginine uptake (Figure 5D; reduction by 46.0 ± 15.4%).More importantly, the reduction of arginine import was associated with a near complete shut-down of proliferation (Figure 5E, reduction by 89.3 ± 8.4%), and an induction of cell death (Figure 5F, increase by 66.4 ± 6.5% of Annexin V-positive cells; Figure 5G, increase by 16.2 ± 7.3% of PI-stained cells) in HG3 CAT-1 k.o. cells grown in normal cell culture medium containing 1 mM arginine.These results were confirmed with HG3 cells, transduced with an alternative CAT-1 shRNA lentivirus (pLKO.1-puro_SLC7A1-1,Supplementary Figure S2).Interestingly, these results are in line with the functional impairment induced in normal, CAT-1-expressing HG3 cells cultured in medium in the complete absence of extracellular arginine (Figures 2A-C).In contrast, downregulation of y + LAT1 or y + LAT2 had no effect on cell proliferation or viability (Supplementary Figure S3).In summary, our data clearly show that CAT-1 is a non-redundant arginine importer for HG3 CLL cells: inhibition of its expression leads to a dramatic impairment of CLL proliferation and also viability. ",
"section_name": "Downregulation of CAT-1 in HG3 Cells Shuts Down Proliferation and Induces Cell Death",
"section_num": null
},
{
"section_content": "We finally wanted to study CAT-1 as a novel potential target for CLL therapy in an in vivo model.To be able to mimic a therapeutic situation, we decided to switch to an inducible CAT-1 knockdown model, in which we could start CAT-1 downregulation at a defined time point.For this model HG3 cells were transduced with a doxycycline-inducible CAT-1 lentiviral SLC7A1-5 shRNA construct and termed iCAT-1 k.o.cells.As control, HG3 cells were transduced with an inducible nontarget shRNA construct and termed iNT cells.iCAT-1 k.o. cells exhibited a pronounced suppression of CAT-1 expression already 4 days after starting doxycycline treatment, and this suppression persisted to the end of the observation period of 21 days (Figure 6A).Like in the constitutive CAT-1 k.o.model (Figure 5E), we saw a strong inhibition of proliferation (Figure 6B; reduction by 82.1 ± 15.1%) and an induction of cell death (Figure 6C: increase by 57.8 ± 13.1% of Annexin V-positive cells; Figure 6D: increase by 15.9 ± 0.4% of PIstained cells) upon doxycycline-mediated CAT-1 knockdown.We then applied these cells to a mouse CLL xenograft: after s.c.injection of HG3 CLL cells (iCAT-1 k.o. or iNT) into the left flank, mice of both tumor groups were split in a treatment group (receiving doxycycline in the drinking water), and a control group (no doxycycline), so that tumor growth was monitored in 4 different experimental groups (Figure 6E).Downregulation of CAT-1 considerably retarded tumor growth as compared to the three control groups in which tumor growth started at very similar time points (Figure 6E).The retardation of tumor growth translated into significantly (p = 0.0035) prolonged survival of mice (Figure 6F).After a pronounced plateau phase with completely inhibited tumor growth, there was in the end breakthrough growth with a growth kinetic that was similar to control tumors (Figure 6E).We studied potential rescue mechanisms in explanted tumors.However, CAT-1 protein expression was persistently downregulated in tumors from iCAT-1 k.o.cell-injected and doxycycline-treated mice (Figure 6G) and the expression of the other arginine transporters was at the same level over all tumor groups (Supplementary Figure S4).In contrast to HG3 cells sufficiently supplied with arginine, ASS was also expressed in tumors with normal CAT-1 expression (Figure 6G). ",
"section_name": "Downregulation of CAT-1 Inhibits CLL Tumor Growth in vivo",
"section_num": null
},
{
"section_content": "Cancer cell proliferation is coupled to the acquisition of carbon and nitrogen, that are mainly derived from non-glutamine amino acids (6), and arginine, carrying four nitrogen atoms, is a precursor for a wide range of nitrogenous compounds (3,10).Targeting nutrient transporter proteins, as the first step fueling the rewired cancer cell metabolism, is a logical, although largely unexplored therapeutic approach (5,44).We therefore investigated arginine transport as a potential new target to treat CLL. We show that CLL cells fulfill the two key requirements, necessary for the successful depletion of arginine by inhibiting one specific transporter: (i) Primary CLL cells in general do not express ASS (Figures 1C,D) and are therefore arginineauxotrophic and (ii) CLL cells non-redundantly rely on the import of arginine via one specific transporter, namely CAT-1 (Figure 5). CLL cells share arginine auxotrophy with other lymphomas (45), acute lymphoblastic leukemia (22) and acute myeloid leukemia (21) as well as with several solid cancer entities (20).Tumor cells shut down ASS expression in order to increase their intracellular availability of the ASS substrate aspartate, which is then shuttled into nucleotide biosynthesis (46).This mostly epigenetically-driven ASS shutdown is on the one side often associated with a more aggressive clinical phenotype (45), on the other side it generates an Achilles heel of cancer cells, that could specifically be targeted by restricting arginine availability (5). Cationic amino acid transport has been described in CLL cells already over 30 years ago (47), although the corresponding transporter was unknown at that time and had not been identified since then.In the current work we have addressed this open question in the context of a renewed interest in cancer cell metabolism and identified CAT-1 as the only arginine importer in both, CLL cell lines as well as PB CLL cells with a strong induction upon TLR9 activation in the latter.Our data are corroborated by recent findings of a coordinately induced network of genes linked to mRNA translation and amino acid metabolism, including various amino acid transporters in both, lymph node (LN) and in TLR9-stimulated PB CLL cells (34). Only few data are published so far on the role of CAT-1 in cancer entities.Its expression has been described in blasts of patients with AML (21) and ALL (22).The siRNAmediated knockdown of CAT-1 in both, breast cancer (48) and colorectal cancer (49) cell lines induced cell death.Upon CAT-1 knockdown, we saw a strong inhibition of CLL cell proliferation in vitro (Figures 5E,6B) and of tumor growth in vivo (Figure 6E) without compensatory upregulation of other potential arginine import proteins or re-expression of CAT-1.The leucine-insensitive arginine uptake was less reduced than the CAT-1 mRNA and protein.This may be due to differences in the proportion of transporter localized in the plasma membrane vs.intracellular compartments.It is remarkable that an about 50% reduction in arginine uptake resulted in the almost complete inhibition of cell proliferation and pronounced cell death.This indicates a very high demand of tumor cells for arginine and suggests either transport inhibition or lowering of extracellular arginine as a promising therapeutic approach.In addition, the pronounced effect of CAT-1 down-regulation on CLL cell viability and tumor growth may partly be due to diminished uptake of the essential amino acid lysine that is also a CAT-1 substrate.However, after initial profound suppression of tumor xenograft growth, there was tumor outgrowth eventually.What might be the reason for this?Our tumor growth curves (Figure 6E) are reminiscent of the temporary arrest of tumor growth in systemically arginine-depleted animals upon application of ADI (50).In ADI-treated patients two prominent resistance mechanisms finally develop: (i) generation of neutralizing antibodies against the microbial ADI enzyme (24), and (ii) re-expression of ASS in cancer tissue with loss of arginine auxotrophy and use of ADI-generated citrulline for endogenous cancer cell arginine synthesis (20).While the former mechanism is irrelevant for our model, the ASS expression indeed detected in tumors of all experimental groups may account for the In physiological cells, arginine may be supplied by uptake via six different transporters (colored red, although not all the transporters listed are expressed within the same cell).The two y + LAT isoforms (colored orange) mediate mostly arginine export by obligatory exchange against neutral amino acids (NAA) plus Na + .Alternatively, arginine can be synthesized in physiological cells from citrulline and aspartate in a two step reaction with argininosuccinate synthase (ASS) as rate-limiting enzyme.In contrast, CLL cells rely solely on the uptake of arginine via CAT-1.Inhibition of CAT-1 is thus a promising new therapeutic approach for CLL and other tumor entities that share the non-redundant dependence on CAT-1 for arginine uptake.AS, argininosuccinate; ASL, argininosuccinate lyase; Asp, aspartate; Cit, citrulline. breakthrough tumor growth (Figure 6E).However, with the exception of CLL cells from one patient, PB primary human CLL cells did not express ASS even under TLR9 stimulation and arginine depletion (Figures 1C,D).We assume that the lack of ASS expression reflects the situation of proliferating CLL cells in vivo, because TLR9 activation induces a specific translational profile mimicking the proliferative compartment of LN CLL cells (34). It remains to be studied how arginine restriction suppresses CLL cell proliferation and viability.In ASS-deficient prostate (51) and breast (52) cancer cells, arginine starvation induces mitochondrial dysfunction-associated autophagic cell death.In breast cancer cells, it has additionally been found that arginine is necessary for arginase II-catalyzed ornithine and subsequent polyamine synthesis to fully execute the cell cycle (53).Arginine deprivation in various ASS-negative tumor cells inhibits the Warburg effect and makes the cells more dependent on glutamine (50).In this model, combination treatment with a glutaminase inhibitor induced synthetic lethality in tumor cells (50).Another way to reduce glutamine availability is interference with its uptake (31, 32, 54-56).We therefore suggest to combine CAT-1 inhibition with strategies to reduce glutamine availability to synergistically induce tumor cell death and prevent the development of metabolic tumor escape mechanisms.Arginine deprivation through CAT-1 inhibition should render tumor cells more vulnerable also to co-applied other anti-cancer therapies.An important question is of course if the fast dividing cells of the immune system would also be affected by such a treatment.We have recently shown that primary human T cells produce endogenously sufficient amounts of arginine from citrulline for unlimited proliferation (18).This suggests that immune cells are protected from a therapeutically induced arginine deficiency.Further studies are needed to confirm a preferential damage of tumor cells by CAT-1 inhibition. Inhibition of amino acid transporters by small molecule compounds as a successful novel anti-cancer strategy (5) has already been reported for SLC1A5 (31), SLC7A5 (56), SLC7A11 (57), and SLC6A14 (58).An appropriate read-out assay for CAT-1 mediated arginine flux (59) could be used for compound library-based high-throughput screening to develop small molecule-or antibody-based inhibitors against CAT-1.These reagents could serve as potential novel cancer therapeutics for CLL and other arginine-auxotrophic tumor entities that share the non-redundant dependence on CAT-1 for arginine uptake (Figure 7). patients/participants provided their written informed consent to participate in this study.The animal study was reviewed and approved by Landesuntersuchungsamt Koblenz. ",
"section_name": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "The authors thank the patients involved in this study for their kind gift of samples; Anders Rosén (Linköping University, Linköping, Sweden) for kindly providing the HG3 cell line, Thorsten Zenz (National Center for Tumor Diseases, Heidelberg, Germany) for kindly providing the MEC1 and JVM-2 cell lines, Dmitri Wiederschain (Novartis Institutes for BioMedical Research, Cambridge, MA) for kindly providing Tet-pLKOpuro vector, Thomas Kindler (University Medicine Mainz) for kindly providing plasmids pLKO.1-puro_SHC002,psPAX2, and pMD2.G and Edite Antunes Ferreira (University Medicine Mainz) for the technical support during mice experiments. ",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
},
{
"section_content": "This work was supported by Deutsche Forschungsgemeinschaft (MU 1547/4-1 and SFB1292/1, 06 to MM; Cl100/6-1to EC), TransMed program of University Medicine Mainz (to AW) and Wissenschaftlicher Verein zur Förderung hämatologischer Forschung e.V. (WVHF 05_2018 and WVHF 07_2015 to AW). ",
"section_name": "FUNDING",
"section_num": null
},
{
"section_content": "The datasets generated for this study are available on request to the corresponding author. ",
"section_name": "DATA AVAILABILITY STATEMENT",
"section_num": null
},
{
"section_content": "The studies involving human participants were reviewed and approved by Rhineland-Palatinate Medical Association Ethics Committee.Blood donors gave written informed consent in accordance with the Declaration of Helsinki.The AW, DP, HE, JR, KR, MT, EC, and MM contributed to the conception and/or design of the work, performed data analysis and interpretation, and revised the manuscript.AW, DP, HE, JR, EC, and MM contributed to the data acquisition.AW, EC, and MM drafted the manuscript.AW, EC, and MM finalized the article.All authors have read and approved the final manuscript and agreed to be accountable for all aspects of the work.This article contains data from the doctoral thesis of DP. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2019.01268/full#supplementary-materialConflict 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. ",
"section_name": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "The studies involving human participants were reviewed and approved by Rhineland-Palatinate Medical Association Ethics Committee.Blood donors gave written informed consent in accordance with the Declaration of Helsinki.The ",
"section_name": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "AW, DP, HE, JR, KR, MT, EC, and MM contributed to the conception and/or design of the work, performed data analysis and interpretation, and revised the manuscript.AW, DP, HE, JR, EC, and MM contributed to the data acquisition.AW, EC, and MM drafted the manuscript.AW, EC, and MM finalized the article.All authors have read and approved the final manuscript and agreed to be accountable for all aspects of the work.This article contains data from the doctoral thesis of DP. ",
"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/fonc.2019.01268/full#supplementary-materialConflict 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. ",
"section_name": "SUPPLEMENTARY MATERIAL",
"section_num": null
}
] |
10.1371/journal.pone.0106773
|
The Peritoneum Is Both a Source and Target of TGF-β in Women with Endometriosis
|
Transforming growth factor-β (TGF-β) is believed to play a major role in the aetiology of peritoneal endometriosis. We aimed to determine if the peritoneum is a source of TGF-β and if peritoneal TGF-β expression, reception or target genes are altered in women with endometriosis. Peritoneal fluid, peritoneal bushings and peritoneal biopsies were collected from women with and without endometriosis. TGF-β1, 2 and 3 protein concentrations were measured in the peritoneal fluid. TGF-β1 was measured in mesothelial cell conditioned media. Control peritoneum and peritoneum prone to endometriosis (within Pouch of Douglas) from women without disease (n = 16) and peritoneum distal and adjacent to endometriosis lesions in women with endometriosis (n = 15) and were analysed for TGF-β expression, reception and signalling by immunohistochemistry, qRT-PCR and a TGF-β signalling PCR array. TGF-β1 was increased in the peritoneal fluid of women with endometriosis compared to those without disease (P<0.05) and peritoneal mesothelial cells secrete TGF-β1 in-vitro. In women with endometriosis, peritoneum from sites adjacent to endometriosis lesions expressed higher levels of TGFB1 mRNA when compared to distal sites (P<0.05). The TGF-β-stimulated Smad 2/3 signalling pathway was active in the peritoneum and there were significant increases (P<0.05) in expression of genes associated with tumorigenesis (MAPK8, CDC6), epithelial-mesenchymal transition (NOTCH1), angiogenesis (ID1, ID3) and neurogenesis (CREB1) in the peritoneum of women with endometriosis. In conclusion, the peritoneum, and in particular, the peritoneal mesothelium, is a source of TGF-β1 and this is enhanced around endometriosis lesions. The expression of TGF-β-regulated genes is altered in the peritoneum of women with endometriosis and this may promote an environment favorable to lesion formation.
|
[
{
"section_content": "Endometriosis is a non-malignant disorder defined by the presence of endometrial tissue outside the uterus, associated with debilitating pelvic pain and impaired fertility [1].The disease is estimated to affect 10-15% of women of reproductive age, with an annual cost recently estimated at $12722, per women [2].Endometriosis is currently managed surgically or medically but symptoms can recur in up to 75% of surgical cases and available medical treatments have undesirable side effects [3,4].A major obstacle in developing new treatment strategies for endometriosis is our limited understanding of the aetiology of this disease. Although shed endometrium and retrograde menses are widely believed to play a role in the aetiology of this enigmatic disorder there is also increasing evidence that the mesothelial cells lining the peritoneal cavity may also contribute to the development and maintenance of peritoneal endometriosis [5].Studies have shown that mesothelial cells provide a surface for ectopic endometrial cell to attach [6][7][8], and it has been reported that they facilitate ectopic endometrial cell invasion through tissue remodelling [9,10] and/or through changes in mesothelial cell morphology [11,12].Perito-neal mesothelial cells secret a wide-range of pro-inflammatory cytokines and growth factors that may promote increased cell proliferation and angiogenesis [10,13].Importantly they may also have impacts on immune cell function, allowing ectopic endometrial cells to evade scavenger cells [14].Notably, a study by Hull et al. showed that peritoneal mesothelial cells, together with other host derived cells, were incorporated into peritoneal-like lesions in a mouse model of endometriosis demonstrating that they may contribute to the cellular constitutes of endometriosis lesions [15]. Transforming growth factors b (TGF-b) is a multifunctional growth factor that is responsible for regulating cell proliferation, differentiation, angiogenesis and immune responses.In the context of the current study these TGF-b mediated processes are all considered to play a role in endometriosis lesion formation [16].There are three TGF-b ligands (TGF-b1, TGF-b2, TGF-b3), which have been shown to have overlapping functions.The ligands are secreted as latent precursor molecules and activated by proteolytic cleavage.Once activated the TGF-bs exert their effects through binding and coupling of TGF-b receptors I and II, which in turn phosphorylate the intracellular factors Smad2 and/or 3 that regulate transcription of target genes [17,18].Dysregulated TGF-b signalling has been implicated in several pathologies and there is growing evidence that TGF-b may play a role in the aetiology of endometriosis.Several studies have reported women with endometriosis to have increased levels of TGF-b in their peritoneal fluid and serum, when compared to women without disease and levels appeared to change across the menstrual cycle [19][20][21].More recently, TGF-b1, and its signalling targets involved in cell survival, were reported to be altered in the eutopic endometrium of women with endometriosis when compared to women without disease [22].Decreased NK cell activity within the peritoneal cavity in women with endometriosis has been attributed to increasing concentrations of TGF-b [23].Interestingly, TGF-b1 was identified as central component of one of four molecular endometrial/peritoneal networks identified in a model of endometriosis and TGFB1 transcripts from both the host (mouse) and endometrial (human) compartments appeared to contribute to lesion development [24].In a follow up study using a mouse model of endometriosis, Hull et al showed TGFB1-null mice, developed fewer and smaller peritoneal endometriosis-like lesions than their wild-type counterparts [15].However, the peritoneum as source of TGF-b has yet to be defined in the context of endometriosis.Furthermore the impacts of increasing TGF-b concentrations on the development and progression of endometriosis remains poorly understood. In this study, we aimed to determine if the peritoneum is a source of TGF-b and if peritoneal TGF-b expression and/or reception are altered in women with endometriosis.We also aimed to investigate the expression profile of key TGF-b target genes in women with and without endometriosis. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "All of the tissues used in this study were collected with informed written consent from women undergoing laparoscopic investigation for chronic pelvic pain, to identify underlying endometriosis under ethical approval obtained from the Lothian Research Ethics Committee (LREC 11/AL/0376).All of the women included in the study were aged between 18-45 years, had regular 21-35 day menstrual cycles and none of them were taking hormonal treatments.The 'control' group of women had no evidence of endometriosis at laparoscopy, nor was there evidence of any other underlying pelvic pathology to explain their painful symptoms (e.g.adhesions).The women with endometriosis had macroscopic evidence of disease at laparoscopy and this was later confirmed by histology. Peritoneal fluid, primary peritoneal mesothelial cells (HPMCs) and peritoneal biopsies were collected at the start of surgery from women with and without endometriosis.Peritoneal fluid (5-10 ml) was collected from women with (n = 6) and without (n = 6) endometriosis and stored in cryovials at 280uC for later analysis.HPMCs (n = 3) were isolated at the time of surgery as previously described by gentle brushing the pelvic mesothelium with a Tao TM brush followed by vigorously agitating in 15 ml of serumcontaining culture media to dislodge cells before transferring to a 75 cm 2 culture flask and incubated at 37uC under 5% CO 2 in air (QC Sciences, Virginia, USA) [25]. In women without endometriosis we biopsied peritoneal tissue (0.5 cm diameter) from sites 'prone' to developing endometriosis lesions, within the Pouch of Douglas (n = 8), and control locations taken 2-3 cm outwith the pelvic brim (n = 13), prone sites were determined in accordance with a study by Mahmood and Templeton 1991 [26].In women with endometriosis, we biopsied peritoneal tissue from sites adjacent to endometriosis lesions, taken 2-3 cm from lesions (n = 3), and at control sites distal to endometriosis lesions, taken 2-3 cm outwith the pelvic brim and in the same position as the control biopsies from women without disease (n = 11).In women with and without endometriosis we collected serum and endometrial biopsies for confirm cycle stage.The tissues were collected according to the Endometriosis Phenome and Biobanking Harmonisation Project (EPHect) guidelines (http://endometriosisfoundation.org/ephect/). Upon collection, peritoneal biopsies were divided into two with half immersed in RNAlater at 4uC for 24 hrs before storage at 2 80uC prior to RNA extraction, and half fixed in 4% neutralbuffered formalin (NBF) for 24 hrs at 4uC before storing in 70% ethanol prior to embedding in paraffin wax for histological studies.All peritoneal biopsies collected were studied histologically to confirm the absence of endometriosis tissue. ",
"section_name": "Subjects",
"section_num": null
},
{
"section_content": "Cycle phase was confirmed: (1) by date of last menstrual period, (2) by the measurement of serum estradiol and progesterone levels, and (3) by examination of endometrial biopsies (stained with haematoxylin and eosin) collected at the same time as the peritoneal fluid samples.The samples were examined by an expert histopathologist who used Noyes' criteria to determine cycle phase.All samples included in this study were in the luteal phase of the cycle. ",
"section_name": "Cycle staging",
"section_num": null
},
{
"section_content": "Brushings of HPMC were cultured in HOSE1 media containing; 40% media 199, 40% MCDB 105 and supplemented with 15% FBS, 0.5% penicillin/streptomycin and 1% L-glutamine, as previously described [25] (Life Technologies Inc., Paisley UK and Sigma Chemical Co., Poole UK).For immunoassay, cells were cultured in serum free conditions for between 12 and 48 hrs.HPMC conditioned culture media was then removed and stored at 280uC for later analysis by immunoassay.Three technical replicates were performed for each patient sample and a total of 3 patient samples were included in this analysis. ",
"section_name": "Establishment of primary human peritoneal mesothelial cell (HPMC) cultures and experimental treatment of HPMC",
"section_num": null
},
{
"section_content": "TGF-b1 ELISA was performed using the Human TGF-b1 Quantikine kit (DB100B), and TGF-b2 and TGF-b3 ELISAs were performed using the Human TGF-b2 (DY302) or Human TGF-b3 (DY243) ELISA Duo set, all according to manufacturers instructions (R&D systems, Abingdon UK).Samples were assayed for active and total TGF-b1-3 levels.For total levels, sampled were pre-treated with 1 M HCL for 10 mins before neutralizing with 1.2 M NaHO/0.5 M HEPES buffer.ELISA plates were read using Lab Systems Multiscan EX Microplate reader at 450 nm with wavelength correction at 540 nm.Samples were quantified using standard curve analysis within the linear range of 2000 pg/ ml to 16 pg/ml.Intra-assay CV was 2.5% and the between batch CV was 8.3% for cell culture supernatants. ",
"section_name": "TGF-b1, TGF-b2 and TGF-b3 Immunoassay",
"section_num": null
},
{
"section_content": "Five micron sections of paraffin embedded tissue were mounted onto electrostatically charged microscope slides and dewaxed in xylene (265min) and rehydrated (VWR, Leicestershire, UK).Antigens were retrieval by pressure-cooking slides in 10 mM Tris 1 mM EDTA pH 9, for 5 mins.Slides were washed in H 2 O before incubation with 3% hydrogen peroxide for 30 min (Sigma).Slides were blocked in either normal horse serum diluted 1:12 in Tris buffered saline with 0.5% Tween 20 (TBST20) or in 5% BSA in Tris buffered saline with 0.5% Tween 20; for 30 mins.Incubation with primary antibody diluted in blocking buffer was performed overnight at 4uC with either; rabbit anti-TGF-b1 (Abcam Ab9758 1:1000), rabbit anti-TGF-bR1 (Santa Cruz 398 1:200), rabbit anti-TGFbR2 (Santa Cruz 220 1:2000), rabbit anti-pSmad2/3 (Santa Cruz 10790 1:1000) or equimolar concentrations of rabbit IgG (Dako X0903) as a negative control (Santa Cruz, Heidelberg, Germany, Abcam, Cambridge UK; Dako, Cambridge, UK and Biosera, Uckfield, UK).Slides were washed in TBST20 and incubated for 30 min with species specific impress kit and washed again before incubation with 3, 39-diaminobenzidine for 5 min (Vector Laboratories).Slides were then counterstained with hematoxylin, dehydrated and visualized by light microscopy, using an Olympus Provis microscope equipped with a Kodak DCS330 camera (Olympus Optical Co., London, UK, and Kodak Ltd., Herts, UK).A total of 8 peritoneal biopsies (n = 2 each group) were examined using immunohistochemistry in total.Due to the limited supply of peritoneal tissue, both positive and negative controls were performed on menstrual stage endometrial tissue. ",
"section_name": "Immunostaining",
"section_num": null
},
{
"section_content": "All peritoneal biopsies assayed for mRNA transcript analysis were from the luteal phase of the cycle.RNA was extracted from peritoneal biopsy tissue using the RNeasy Mini Kit with oncolumn DNase1 digestion (Qiagen, Manchester UK).First-strand cDNA synthesis was performed using Superscript VILO Master Mix (Life Technologies Inc.).Both processes were performed according to the manufacturer's instructions. Quantitative (q)RT-PCR was performed with brilliant III ultrafast SYBR green qRT-PCR master mix using an ABI Prism 7900 Fast system under standard running conditions with pre-validated primers shown in Table 1 (Applied Biosystems, Warrington, UK, Agilent, Berkshire, UK and Primerdesign).Messenger RNA transcripts for TGF-b1 (TGFB1), TGF-bR1-2 (TGFBR1 TGFBR2) and Smad 3 (SMAD3) were quantified using the 2 2DCt method relative to GAPDH, as determined by geNorm housekeeping assay (Primerdesign). ",
"section_name": "mRNA transcript analysis",
"section_num": null
},
{
"section_content": "The PCR array plate included 84 TGF-bregulated genes involved in functional processes such as differentiation, proliferation, migration, apoptosis and cell cycle control.All peritoneal biopsies assayed in the TGF-b signalling PCR array were matched samples from the luteal phase of the cycle.RNA was extracted from peritoneal biopsy samples from women without endometriosis at control sites (n = 3) and sites prone to endometriosis (n = 3) and from women with endometriosis at sites distal (n = 3) and adjacent (n = 3) to endometriosis lesions.RNA was adjusted to 125 ng/ml and reverse transcribed using the RT 2 single strand cDNA synthesis kit according to manufacturers instructions (Qiagen).RT-PCR reactions were prepared using RT 2 qPCR master mix using an ABI Prism 7900 Fast system according to the manufacturer's instructions.Results were quantified using the web portal: http://www.SABiosciences.com/pcrarayanalysis.php. ",
"section_name": "TGF-b signalling PCR array",
"section_num": null
},
{
"section_content": "All the results are expressed as mean 6 standard error of the mean of a minimum of 3 independent experiments.ELISA data was analyzed using unpaired students t test after testing for normal distribution.Quantitative RT-PCR was analyzed using paired and unpaired students t tests, as appropriate after testing for normal distribution.All statistical results were generated using GraphPad PRISM version 5 statistical software (GraphPad Software Inc, San Diego, USA) and a P value of ,0.05 was considered significant. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "We assayed active and total concentrations of TGF-b1, TGF-b2 and TGF-b3 in the peritoneal fluid of women with and without endometriosis in the luteal phase of the menstrual cycle.Active concentrations of TGF-b1-3 were undetectable in peritoneal fluid; therefore we used total concentrations only in this analysis.We found all TGF-b ligands to be present in the peritoneal fluid of women with and without endometriosis (Figure 1A).TGF-b1 was significantly increased in the peritoneal fluid of women with endometriosis when compared to women without (Figure 1A).We found no significant changes in TGF-b2 or TGF-b3 levels between women with and without endometriosis (Figure 1A).To determine if the peritoneum is source of TGF-b1, we measured TGF-b1 protein in conditioned media from primary cultures of HPMCs (Figure 1B).We found HPMCs to secret TGF-b1 protein and this appeared to increase over time, although no significant difference was found with the time points analysed (Figure 1B).TGF-b1 and its receptors together with phosphorylated Smad 2/3 are localized to the mesothelial cells We next examined peritoneal biopsies for presence and localization of TGF-b1 by immunohistochemistry. TGF-b1 was present in peritoneum from women with and without endometriosis and was localized to peritoneal mesothelial cells (Figure 2).To investigate whether the peritoneum is receptive to TGF-b signalling, peritoneal biopsies from women with and without endometriosis were examined for presence and localization of TGF-b receptors 1 and 2 and phosphorylated Smad 2/3 (pSmad 2/3) by immunohistochemistry. TGF-b receptors 1 and 2 and pSmad 2/3 (Figure 2) were localized to the peritoneal mesothelial cells of peritoneal biopsies from women without disease (Figure 2) and women with disease (Figure 2).There was no staining observed in the isotype matched control sections (Figure 2).The peritoneum adjacent to endometriosis lesions expressed increased TGFB1 To investigate if the peritoneal source of TGF-b1 or its signalling components, TGF-bR1, TGF-bR2 and Smad3, are altered in women with endometriosis, we quantified mRNAs encoded by TGFB1, TGFBR1, TGFBR2 and SMAD3.Notably TGFB1 mRNAs were significantly increased (p,0.05) in peritoneum adjacent to endometriosis lesions when compared to peritoneum that was distal to the lesion (Figure 3B), but there was no change in TGFB1 expression between women with endometriosis and those without (Figure 3C) or between sites of peritoneum in women without disease (Figure 3A).There was no significant changes in expression of TGFBR1, TGFBR2 or the downstream regulator SMAD3 in all comparisons made (Figure 3 A, C-L), suggesting that responsiveness to TGF-b ligands is maintained adjacent to the lesion. ",
"section_name": "TGF-b1 concentrations are increased in the peritoneal fluid of women with endometriosis and the peritoneal mesothelium synthesizes and secretes TGF-b1",
"section_num": null
},
{
"section_content": "We investigated the expression of TGF-b signalling targets in peritoneal biopsies from women with and without endometriosis in the luteal phase of the menstrual cycle, using a commercial TGF-b target PCR array.We made three comparisons using this methodology.Firstly, we compared peritoneum from sites prone (n = 3), to control sites (n = 3), to developing endometriosis, in women without endometriosis.As endometriosis is most commonly found within the pelvic brim, we made this comparison to determine if there was any difference in the peritoneal tissue at this location that may predispose to the development of endometriosis [26]. Secondly, we compared peritoneum from women with endometriosis from sites adjacent (n = 3) and distal (n = 3) to endometriosis lesions.We made this comparison to determine if there was any difference in the peritoneal tissue at this location that may contribute to the development of endometriosis. Finally, we compared peritoneum from women with endometriosis at sites adjacent to endometriosis lesions (n = 3) to peritoneum from women without disease at sites prone to endometriosis (n = 3).We made this comparison to determine if there was any difference in the peritoneal tissue between women with and without endometriosis. The overall expression profile for all array candidate genes can be found in Data S1.Of the 82 genes analysis, we found 3 genes (AIPL1, MYOD1, SHH), to be undetectable in all samples.Only one gene, IL10, was significantly increased at the sites prone to endometriosis when compared control peritoneal (fold change 2.75, p = 0.026) (Figure 4). When we compared peritoneum from women with endometriosis to peritoneum from women without we found six genes to have significant changes in expression (Table 2, Figure 4).These genes included NOTCH1 involved in cell development and TGFb induced EMT [27] and CREB1 associated with neurogenes [28].ID1 and ID3, associated with cell differentiation, angiogenesis and reported to be increased in several cancers [29]; MAPK8 associated with cell cycle control and cell differentiation [30] and CDC6 a protein involved in DNA replication and which has been implicated in EMT and cancer development [31]. Finally we found four genes to be significantly increased in the peritoneum adjacent to endometriosis lesions in women with endometriosis compared to peritoneum distal to the lesion (Table 3, Figure 4).These were NOTCH1 and CREB1 as previously mentioned; SOX4 a transcription factor that is increased during EMT and heavily associated with tumorigenesis and metastasis [32] and PTK2B essential in neuropeptide signalling [28]. ",
"section_name": "The expression of several TGF-b-regulated genes is increased in peritoneum adjacent to endometriosis lesions",
"section_num": null
},
{
"section_content": "In this study, we have demonstrated that TGF-b1 is increased in the peritoneal fluid of women with endometriosis, and that TGF-b1 and TGF-b signalling targets associated with cell cycle control, angiogenesis, EMT and tumorigenesis are significantly increased in the peritoneum of women with endometriosis compared to those without.We believe that these changes may help establish an environment favourable for endometriosis lesion formation within the peritoneum contributing to the aetiology of this disease. Peritoneal fluid levels of TGF-b1 is significantly increased in women with endometriosis during the luteal phase.These results are in agreement with several studies reporting TGF-b and TGF-b1 levels to be increased in women with endometriosis and also to be increased across the menstrual cycle [19][20][21].In only one pervious study [33] did the authors indicate if total or bioactive levels of TGF-b were measured, another did not differentiate between the TGF-b ligands assayed [34].In the current study we have extended these findings reporting bioactive TGF-b1, 2 and 3 are undetectable in the peritoneal fluid, suggesting that they may only have an effect following activation within the peritoneum or lesions.A previous study examined activation of TGF-b in women with endometriosis via the plasminogen activation pathway that was found to be increased at sites of endometriosis lesions [35]. In this study, we demonstrated that the HPMC secreted TGF-b1 protein and these cells may contribute to the significant rise in TGF-b1 in the peritoneal fluid of women with endometriosis.Peritoneal mesothelial cells are known to overexpress TGF-b1 ligands into the peritoneal fluid in response to peritoneal inflammation [36] and this has been linked to pathologies such as peritoneal fibrosis and cancer.Furthermore, this increase in TGF-b1 ligand expression may have direct effects on adjacent endometriotic cellular activity.For example, TGF-b1-null mice had significantly reduced numbers of macrophages within endometriosis-like lesions, when compared to wild-type mice, suggesting that TGF-b1 plays a critical role in macrophage recruitment to lesions [15].Furthermore, decreased natural killer cell activity within the peritoneal cavity in women with endometriosis has been attributed to increasing concentrations of TGF-b within the peritoneal fluid [23].To date, it is not yet known if the increased levels of peritoneal fluid TGF-b1 precedes or follows the development of endometriosis.However, as retrograde menstruation and the presence of endometrial cells within the peritoneal cavity can induce inflammation, the development of endometriosis and the increase in TGF-b1 are likely to go hand-in-hand [37]. We have also shown that the peritoneum from women with endometriosis expressed higher levels of TGFB1 mRNA transcripts in peritoneum adjacent to endometriosis lesions, when compared to peritoneum distal to endometriosis lesions.This suggests that there is a local disruption of the peritoneum signalling pathways and may indicate that the peritoneum adjacent to endometriosis lesions plays a part in their establishment and survival leading to an integrated microenvironment.We found no difference in expression of TGFB1 neither between women with and without endometriosis nor between sites of peritoneum in women without endometriosis.However, whether women with endometriosis have an altered TGF-b peritoneal environment that predisposes to endometriosis, or, that the presence of peritoneal endometriosis lesions induce changes in adjacent peritoneum and the peritoneal fluid is not clear.Given that expression of TGFB1 is significantly different in peritoneum from sites distal to endometriosis lesions compared to sites adjacent to endometriosis lesions but there is no difference in peritoneal expression of TGFB1 between women with and without endometriosis lesions, it is likely to be the later and not a global change in the peritoneum of women with endometriosis.This increase in TGFB1 expression may be a result of the increased inflammatory environment induced by the presence of an endometriosis lesion. Interestingly, we have shown that the peritoneum is responsive to TGF-b signalling but that there was no change in TGF-b receptor expression or Smad 3 expression, suggesting that the sensitivity of the peritoneum to TGF-b remained similar in all comparisons made.Therefore, increasing TGF-b1 levels in women with endometriosis is likely to have significant down stream effects on TGF-b signalling targets within peritoneal tissue. Lastly, we assessed expression of TGF-b signalling targets in the peritoneum of women with and without endometriosis.We compared peritoneum from women without endometriosis and looked at expression levels between sites of peritoneum prone to developing endometriosis compared to control peritoneum.We found only one gene, IL10 coding for cytokine interluken-10 (IL-10), to be significantly increased.IL-10 is reported to be increased in the peritoneal fluid and serum of women with endometriosis and increasing IL-10 has been linked to a decrease in CD4+ T lymphocyte activation and therefor decreased immune responses [38,39].A mouse model of surgically induced endometriosis demonstrated that IL-10 promoted the growth of endometriosis lesions and this was suggested to be due to IL-10 supressed immunity allowing endometrial implants to develop [38].However as we found no differences in TGF-b expression or signalling within this tissue type comparison, changes in IL10 expression may not be TGF-b dependant.Nevertheless an increase in IL-10 expression in peritoneum within the pouch of Douglas may lead to supressed immunity within this local area allowing for ectopic endometrial cells to survive and develop into endometriosis lesions. When we compared peritoneum from women with endometriosis to women without endometriosis, we saw a significant change in six genes.The significant changes in the expression of TGF-b signalling targets co-insides with the observation that there is significantly higher TGF-b1 within the peritoneal fluid of women with endometriosis.Of those increased, NOTCH1 is associated with cancer metastasis though TGF-b induced EMT and EMT of endometriotic cells has recently been implicated in the development of endometriosis [40,41].This data supports the hypothesis that the peritoneum of women with endometriosis may also be undergoing EMT and this could help facilitate ectopic cell invasion [5].Transcription factor CREB1 a key regulator of neurotropic factors such as nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF) was also significantly increased.NGF and BDNF have recently been described in the context of endometriosis associated pain and regulation of neurogenesis in and around the sites of lesions [28]. ID1 and ID3 share a similar pattern of expression and both were significantly increased in the peritoneum of women with endometriosis.ID1 and ID3 are essential for angiogenesis and neurogenesis during development and tumor growth, with ID1 +/2 ID3 2/2 knockout mice unable to support the growth of tumors [29].Overexpression of ID1 has been described in several cancers and has been specifically linked to increases in VEGFA gene transcription [42][43][44].Increased VEGF expression is essential for endometriosis lesion development through the initiation of angiogenesis [45][46][47].However a role for ID proteins in the aetiology of endometriosis has not been reported. MAPK8 that plays a critical role in proliferation, differentiation and apoptosis and MAPK8 activity has been implicated in a range of human diseases including cancer, neurological and inflammatory conditions [30].Two studies have demonstrated MAPK8 activity in endometriosis and this was linked to increased inflammatory responses [48,49].MAPK8 has been shown to inhibit apoptosis though Bad which can also induce glycolysis, a common trait seen in nearly all cancers [50][51][52].This supports findings from a recent study from our laboratory that demonstrated that there are metabolic changes in the peritoneum of women with endometriosis, consistent with the induction of glycolysis, and which may promote the establishment and progression of endometriosis lesions [53].CDC6 was also significantly increased.CDC6 is an ATPase associated with cell cycle progression but has not been described in the pathophysiology of endometriosis [31]. Subsequently, we assessed changes in women with endometriosis from sites adjacent to endometriosis compared to sites distal to endometriosis lesions.We found three genes to be significantly increased and one gene to be significantly decreased.NOTCH1 and CREB1 as described above, were significantly increased suggesting that the peritoneum adjacent to endometriosis lesions may be more prone to undergoing TGF-b induced EMT and neurogenesis [27], [40,41].Transcription factor SOX4 was increased in peritoneum adjacent to endometriosis lesions.SOX4 is reported to have impacts on inhibition of apoptosis, increased cell proliferation and EMT, although a role for SOX4 has not been described in the context of endometriosis [32].Together these results suggests that there are local environmental changes in the peritoneum that surrounds endometriosis lesions that are similar to those seen in tumorigenesis and that these changes may help facilitate endometriosis development and progression. In this study, we have shown that the peritoneal mesothelium may be responsible for the increased TGF-b levels in women with endometriosis.Furthermore we have shown that expression of several TGF-b signalling targets is altered within the peritoneum of women with endometriosis and these may play a role in lesion development.These data suggest that the peritoneum expresses higher levels of genes associated with tumorigenesis, inflammation and EMT indicating that the peritoneum may contribute to the development of endometriosis by facilitating growth and survival through angiogenesis, cell invasion through EMT and may play a role in endometriosis associated pain though neurogenesis.These data shed new light on how TGF-b signalling plays a significant role in endometriosis and provides new targets that may be beneficial for the management of endometriosis. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "We are grateful for the help of Mrs Helen Dewart and Mrs Ann Doust for patient recruitment and sample collection; Ms Abby Dobbins, Mr Bob Morris, Mrs Frances Collins, Ms Arantza Esnal-Zufiurre and Mrs Jean Wade for technical support and advice; Mrs Sheila Milne for secretarial support; Mr Ronnie Grant and Mr Jeremy Tavener for graphics support. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "This work was funded by a Wellbeing of Women research grant (R42533) awarded to AWH, PTKS, JKB and WCD (http://www.wellbeingofwomen.org.uk).VJY receives grant support from Federation of Women Graduates (134225) and a PhD studentship from the College of Medicine and Veterinary Medicine at the University of Edinburgh (http://www.bfwg.org.uk).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": "The authors confirm that all data underlying the findings are fully available without restriction.All relevant data are within the paper and its Supporting Information files. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Data S1 The TGF-b signalling targets array (Quigen, Manchester UK) assays 84 TGF-b regulated genes involved in functional processes, including; differentiation, proliferation, migration, apoptosis and cell cycle control.We made three comparisons using this methodology.Firstly we compared peritoneum from sites prone to developing endometriosis to control sites of peritoneam, in women without endometriosis.Secondly we compared peritoneum from women with endometriosis from sites adjacent and distal to endometriosis lesions.Finally we compared peritoneum from women with endometriosis at sites adjacent to endometriosis lesions to peritoneum from women without disease at sites prone to endometriosis.The overall expression profile for all functionally focused genes assayed is listed here.Genes are grouped according to functionality as listed by the manufacturer in the product specification literature.Due to several genes having a wide functionality, gene responses may be listed in more than one table .(DOCX) ",
"section_name": "Supporting Information",
"section_num": null
},
{
"section_content": "",
"section_name": "Author Contributions",
"section_num": null
}
] |
10.2119/2008.00061.mccarthy
|
Surface Expression of Bcl-2 in Chronic Lymphocytic Leukemia and Other B-Cell Leukemias and Lymphomas Without a Breakpoint t(14;18)
|
Since its discovery in follicular lymphoma cells at the breakpoint t(14;18), Bcl-2 has been studied extensively in many basic and clinical science settings. Bcl-2 can locate as an integral mitochondrial membrane component, where its primary role is to block apoptosis by maintaining membrane integrity. Here we show that Bcl-2 also can position on the outer cell surface membrane of B cells from patients with chronic lymphocytic leukemia (B-CLL) and certain other leukemias that do not classically possess the chromosomal breakpoint t(14;18). Although low levels of Bcl-2 can be detected on the surface membrane of apparently healthy leukemic and normal B cells, expression of Bcl-2 correlates best with spontaneous or induced apoptosis. Notably, upon induction of apoptosis, B-CLL cells were much more efficient in upregulating surface Bcl-2 than normal B cells. It is not clear if this surface membrane expression is a passive consequence of the apoptotic process or an active attempt by the B cell to abort cell death by stabilizing the plasma membrane.
|
[
{
"section_content": "Bcl-2 was identified initially at a common chromosomal breakpoint in B-cell follicular lymphomas t(14;18) (1).This seminal finding led to the discovery of a family of ~20 proteins involved in the regulation of programmed cell death (reviewed in [2]).The Bcl-2 family contains both anti-and pro-apoptotic members, and the balance of these counteracting forces is crucial to the fate and expansion of normal and neoplastic B lymphocytes.Bcl-2 can play a primary role in oncogenesis by inhibiting apoptosis, and was the first member of a new category of oncogenes: regulators of cell death (3). The anti-apoptotic members of the Bcl-2 family are involved in checkpoints upstream of mitochondrial dysfunction and caspase activation (3).By virtue of its pivotal position in the life and death cycle of a cell, Bcl-2 may be an excellent candidate for therapeutic intervention (4,5).Early clinical trials utilizing an antisense strategy to alter Bcl-2 levels have shown some encouraging initial results (6); additional promising results were reported recently using small molecule in-hibitors of Bcl-2 function, such as the BH3 mimetic ABT-737 (7). After synthesis, Bcl-2 is chaperoned to several intracellular sites by FKBP38 (8), an FK506-binding immunophilin protein (9).At these sites, Bcl-2 becomes integrated into the cytoplasmic face of the intracellular membranes by its carboxyl-terminal, hydrophobic 17 amino acid tail (10).Bcl-2's role, when positioned at the mitochondrial surface, is to maintain organelle integrity and prevent apoptosis (11,12).In addition, the molecule is associated with the cytoplasmic surfaces of the endoplasmic reticulum (ER) and the nuclear envelope (13).Paradoxically, when localized to the nuclear compartment, Bcl-2 can induce apoptosis by inhibiting transcription factor transport into the nucleus, thus highlighting that cellular sublocalization can determine opposing actions (14). ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "Leukemia and Other B-Cell Leukemias and Lymphomas Without a Breakpoint t (14;18) Since its discovery in follicular lymphoma cells at the breakpoint t(14;18), Bcl-2 has been studied extensively in many basic and clinical science settings.Bcl-2 can locate as an integral mitochondrial membrane component, where its primary role is to block apoptosis by maintaining membrane integrity.Here we show that Bcl-2 also can position on the outer cell surface membrane of B cells from patients with chronic lymphocytic leukemia (B-CLL) and certain other leukemias that do not classically possess the chromosomal breakpoint t (14;18).Although low levels of Bcl-2 can be detected on the surface membrane of apparently healthy leukemic and normal B cells, expression of Bcl-2 correlates best with spontaneous or induced apoptosis.Notably, upon induction of apoptosis, B-CLL cells were much more efficient in upregulating surface Bcl-2 than normal B cells.It is not clear if this surface membrane expression is a passive consequence of the apoptotic process or an active attempt by the B cell to abort cell death by stabilizing the plasma membrane.In this manuscript, we demonstrate the presence of Bcl-2 on the surface membrane of human B lymphocytes, especially leukemic B cells from patients with B-cell type chronic lymphocytic leukemia (B-CLL) and certain related diseases.Although the function of cell surface-associated Bcl-2 is not clear, its appearance, primarily on cells undergoing apoptosis, suggests a relationship between surface membrane re-localization and the apoptotic process. ",
"section_name": "Surface Expression of Bcl-2 in Chronic Lymphocytic",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "The Institutional Review Board of the North Shore-LIJ Health System approved these studies.Following informed consent obtained in accordance with the Declaration of Helsinki, peripheral venous blood was taken from B-CLL patients and healthy subjects.B-CLL clones expressing IGHV genes differing by ≥ 2% from the most similar germline gene were defined as Mutated-CLL (M-CLL), and clones expressing IGHV genes with < 2% difference from germline gene as Unmutated-CLL (U-CLL). For gene expression profiling studies, peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Hypaque (Ficoll-Paque; Pharmacia LKB Biotechnology, Piscataway, NJ, USA) density gradient centrifugation, and then B cells were isolated by negative selection using the B-cell Isolation Kit (Miltenyi, Auburn, CA, USA) and MScolumns.For real-time quantitative polymerase chain reaction (RT-QPCR) verification, freshly isolated B-cells from B-CLL patients and control subjects matched for age (≥ 55 years) were separated by density gradient centrifugation and isolated by negative selection using RosetteSep (StemCell Technologies, Vancouver, Canada) according to the manufacturer's protocol.In some cases, particularly other malignancies, PBMC were cryopreserved with DMSO using a programmable cell-freezing machine (Cry-oMed, Mt.Clemens, MI, USA).These were subsequently thawed and analyzed. In both cases, total RNA was isolated with the RNeasy kit (Qiagen, Valencia, CA, USA) as suggested by the manufacturer.For microarray analysis, double stranded cDNA was synthesized from total RNA using a T7-polyT primer and the SuperScript Choice System Kit (GibcoBRL/Invitrogen Carlsbad, CA, USA).cDNA was purified by phenol/ chloroform extraction and biotinylated by in vitro transcription using the Bio Array High Yield RNA transcript labeling kit (ENZO, Farmingdale, NY, USA).cRNA was purified and DNAse treated using the RNeasy kit, fragmented according to the Affymetrix protocol, and 15 µg hybridized to the Human HG-U95 or Human HG-U133 microarray chip sets (Affymetrix, Santa Clara, CA, USA).Chip hybridization, washing, and imaging were performed by the Core Facility of The Feinstein Institute for Medical Research following Affymetrix protocols and analyzed using Affymetrix-provided software.We used .celand .txtfiles thus generated for subsequent analyses with Microsoft Excel (Redmond, WA, USA), DChip (Boston, MA, USA), GeneCluster (Cambridge, MA, USA), and Genesifter (Seattle, WA, USA). ",
"section_name": "Patients and Healthy Donors",
"section_num": null
},
{
"section_content": "Freshly isolated and negatively selected (RosetteSep) human B-cells, both from CLL patients and normal subjects, were cultured in IMDM media (Glutamax, GIBCO, Carlsbad, CA, USA) supplemented with 5% human AB serum (Atlanta Biologicals, Lawrenceville, GA, USA), 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol (Chemicon, Temecula, CA, USA), and penicillin (100 U/mL)streptomycin (100 µg/mL) (GIBCO-BRL Life Technologies; Grand Island, NY, USA).In addition, the following cell lines were used: MEC-1 (15); RAMOS (Burkitt's lymphoma, IgM); Dakiki (Epstein-Barr virus-transformed lymphoblast, IgA), Jurkat (acute T-cell leukemia); and 697 (acute lymphoblastic leukemia) (697 was obtained from DSMZ [Braunschweig, Germany]).697-Bcl-2 cells were verified to have a 10-fold higher expression of Bcl-2 relative to 697-Neo cells (data not shown).697 cells were maintained in RPMI 1640 supplemented with 10% human serum, plus glutamine and antibiotics.Other cell lines were maintained in RPMI medium (GIBCO) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL)streptomycin (100 µg/mL) (GIBCO-BRL), and 2 mM L-glutamine (GIBCO-BRL).All cells were cultured in humidified air with 5% CO 2 at 37°C.Cells were induced to undergo apoptosis by exposure to camptothecin (Sigma, St Louis, MO, USA) or by changes in culture temperature.In the latter case, cells were placed in an Eppendorf tube and immersed in a water bath or placed on a heat block for 3-5 min at either 45°or 60°C before or after staining.Although either order yielded similar results, optimal data were obtained when cells were surface stained and then heated with a heat block.Flow cytometric gating was based on the isotype control antibody provided by the manufacturer and the amount of nonspecific signal in cells singly stained with Annexin V. ",
"section_name": "Cell Culture",
"section_num": null
},
{
"section_content": "Cells were collected and washed with cold PBS, erythrocytes removed with lysis buffer containing protease inhibitors (both from Roche, Basel, Switzerland) and stored at -20°C.Next, 5 mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.1% sodium deoxycholate containing 1:40 dilution of protease inhibitor cocktail for mammalian cells and 1:50 dilution of phosphatase inhibitor cocktail 2 (both from Sigma-Aldrich, St. Louis, MO, USA) were added.The protein concentration of each cell lysate was determined with the RC and DC Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred on Immobilon-P polyvinylidene difluoride membranes (Millipore, Bradford, MA, USA).Thirty µg of protein were loaded into each lane. Membranes were blotted at 4°C overnight with mouse anti-human Bcl-2 (BD, Franklin Lakes, NJ, USA).Immunodetection was achieved with goat antimouse IgG horseradish peroxidase (HRP)-linked polyclonal antibodies (New England BioLabs, Ipswich, MA, USA) and the ECL Plus (Enhanced Chemiluminescence) detection system (Amersham Biosciences, Piscataway, NJ, USA) with BioMax MR films (Eastman Kodak, Rochester, NY, USA).Protein bands were analyzed by ImageQuant software (Amersham Biosciences). ",
"section_name": "Western Blot Analyses",
"section_num": null
},
{
"section_content": "The relative expression of mRNA was determined using the ABI PRISM 7700 Sequence Detection System and TaqMan chemistry (Applied Biosystems, Foster City, CA, USA).Briefly, a set of forward and reverse primers were designed with a probe labeled with a reporter at the 5'end and a quencher at the 3'end in an internal region of the target sequence.Fluorescence was directly proportional to the amount of target RNA.Fluorescent intensity was measured at every cycle, and cycle numbers were determined at a point in which every sample was in the exponential phase.All samples were analyzed in duplicate, and β-actin was used as an internal control gene.Results were obtained as Ct (Threshold cycle) values, in which Ct is inversely proportional to the starting template copy number.Relative expression of the target gene was calculated in comparison to untreated control samples using the delta delta Ct method (User Bulletin #2; Applied Biosystems).Results were expressed as fold change with respect to the experimental control set being normalized to one.Primer sequences: Forward Primer: AAATCCATGCACCTAAACCTTTTG; Reverse: CAAATTCTACCTTGGAGGGA AAAAAC; Taqman Probe: CCGTG GGCCCTCCAGATAGCTCAT. ",
"section_name": "RT-QPCR",
"section_num": null
},
{
"section_content": "Freshly isolated or thawed, negativelyselected B cells from B-CLL patients and normal healthy donors were washed with PBS containing 1% FBS and 0.1% sodium azide, and incubated for 20 min at 4°C with one of two mAbs (mouse 100 or hamster 6c8) specific for Bcl-2 (BD), with and without lineage specific antibodies to CD19, CD5, and CD11b.After fixation in 1% formaldehyde, cells were compared with others, similarly prepared but exposed to fluorochrome-conjugated isotype control mAbs, by flow sorting using a FACS-Aria (BD Biosciences, San Jose, CA, USA) or a FACS-Caliber (BD Biosciences).Cryopreserved samples from patients with acute lymphoblastic leukemia (ALL), hairy cell leukemia (HCL), and multiple myeloma (MM) were analyzed after costaining with mAbs specific for CD10, CD19, and CD138, respectively.Cells from untreated B-CLL patients and normal subjects were gated on the lymphocyte group prior to analysis; other untreated leukemias were gated on singlets.Experimental samples were analyzed as total cells or gated to eliminate debris. Cell cycle changes and the occurrence of apoptosis were detected by flow cytometry after incubating cells with propidium iodide (PI; Calbiochem, San Diego, CA, USA). 1 × 10 6 cells were exposed to hypotonic PI (0.05 mg/mL PI, 0.1% Triton X-100), and data were acquired on a FACS-Caliber with a 488-nm argon laser.In some instances, cells were examined for viability with Annexin V conjugated to FITC, PE, or APC (BD Biosciences).Acquisition and analysis were performed with CellQuest software (BD Biosciences) of FlowJo (Tree Star Inc, Ashland, OR, USA).Apoptotic cells in PI samples were calculated from the subG 1 fraction as previously described (16,17). ",
"section_name": "Flow Cytometry",
"section_num": null
},
{
"section_content": "Cells were washed and stained with appropriate mAbs as described for flow cytometry and then exposed to 4% paraformaldehyde for 20 min, followed by another wash.Anti-fade reagent (ProLong Gold; V, Carlsbad, CA, USA) was added before mounting on slides with coverslips.Isotype control mAbs were used to evaluate non-specific binding.Images were collected using a 60 × numerical aperture 1.4 PlanApo infinity-corrected objective with an Olympus Fluoview 300 confocal microscope (Olympus America Inc., Center Valley, PA, USA).Files were converted to bitmap in Paint (Microsoft, Redmond, WA, USA) and adjusted for brightness and contrast with Microsoft Picture Manager (Microsoft). ",
"section_name": "Confocal Microscopy",
"section_num": null
},
{
"section_content": "Data were analyzed with Microsoft Excel for statistical significance using Student t test.Two-tailed distributions were used except for experiments in which apoptosis was induced with camptothecin (Sigma, St Louis, MO, USA) or by heat, for which a one-tailed t test was utilized, since these treatments would not be expected to increase viability.P values < 0.05 were considered statistically significant.Error bars represent standard deviations. All supplementary materials are available online at molmed.org. ",
"section_name": "Statistical Analyses",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "B-CLL cells have enhanced levels of intracellular Bcl-2 (18), thereby assisting the tumor cells in evading elimination by apoptosis.We verified these findings, at the RNA level, by microarray analyses with two Affymetrix chips followed by RT-QPCR, and, at the protein level, by intracellular immunofluorescence and Western blotting (Figure 1).Upon initial screening based on Affymetrix probe calls, 6 of 13 microarray probe sets showed highly significant increases in Bcl-2 mRNA levels.The average fold change for B-CLL patients (n = 35) compared with controls (n = 24) was 4.44 (Figure 1B), which correlated well with the average RT-QPCR fold change for 18 paired B-CLL and normal samples, 5.53 (Figure 1C). Western blotting analyses comparing B-cell lysates of five B-CLL patients with those of three normal individuals matched for age also confirmed increased levels of Bcl-2 protein in all leukemic samples relative to control (Figure 1D).For the cohort of B-CLL samples used in Figure 2, an average of 98.45% of cells expressed intracellular Bcl-2 by immunofluorescence (data not shown). ",
"section_name": "Bcl-2 is Elevated in B-CLL at the RNA and Protein Levels",
"section_num": null
},
{
"section_content": "We exposed freshly drawn, never frozen B cells from U-CLL (n = 19) and M-CLL (n = 20) patients to FITC-labeled mAbs specific for Bcl-2 (see Figure 2, Supplemental Figure 1) and analyzed for surface membrane (sm) expression of the protein by flow cytometry.Leukemic B cells clearly expressed smBcl-2 (Figure 2A).This was the case for both M-CLL and U-CLL clones, and there was no statistically significant difference in smBcl-2 levels between the two patient subgroups (P = 0.65).SmBcl-2 expression by B-CLL cells was verified with the same specific mAb linked to a different fluorochrome, PE; furthermore, analyses with another mAb clone isolated from a different species, Armenian hamster, yielded similar results (data not shown). The pattern of smBcl-2 expression varied among B-CLL clones, ranging from distinct, dense populations of CD19 + CD5 + smBcl-2 + cells (Figure 2B, far right column) to CD19 + CD5 + smBcl-2 + populations containing fewer cells (Figure 2B, second column from right).Finally, a subset of clones expressed only a few CD19 + CD5 + smBcl-2 + cells (Figure 2B, third column from right ). ",
"section_name": "Bcl-2 Detected on the Surface Membrane of B-CLL Cells",
"section_num": null
},
{
"section_content": "We next evaluated smBcl-2 expression on fresh human B lymphocytes from healthy, age-matched subjects.Normal B cells expressed smBcl-2, albeit at levels less than that of B-CLL cells (Figure 2A,2B).This difference in membrane expression was highly significant when compared with B-CLL as a whole (P = 3.75 × 10 -6 ; see Figure 2A) or with its subtypes (U-CLL, P = 8.26 × 10 -5 and M-CLL, P = 5.05 × 10 -6 ). In addition, we analyzed smBcl-2 expression on human cord blood B cells.These cells were chosen because of surface membrane expression of CD5, a characteristic marker for CLL (19,20).Cord blood expression of smBcl-2 was not of the same magnitude as seen in B-CLL, as evidenced by the insignificant P value for the CD5 + (P = 0.07) as well as CD5 -(P = 0.21) cord blood cells.Although smBcl-2 expression was more evident among CD5 + cord blood cells (n = 5) than CD5 - cord blood cells (n = 7) from the same samples (2.39% -5.07%; see Figure 2A), this difference was not significant. Several cell lines also were analyzed.Dakiki, an EBV transformed, IgAexpressing B-cell line from a patient with nasopharyngeal carcinoma; Jurkat, a T lymphoblast cell line from a patient with acute T cell leukemia; and MEC1, a transformed B-cell line derived from a B-CLL patient, were each negative for smBcl-2 (data not shown).Similarly, a human acute lymphoblastic leukemia cell line, 697, which was transfected with Bcl-2, did not have detectable smBcl-2, before or after transfection.RAMOS B cells, a Burkitt's lymphoma cell line, showed some positive cells, particularly after apoptotic induction by irradiation, although a large amount of non-specific binding occurred after apoptosis induction (data not shown). ",
"section_name": "Bcl-2 Detected on the Surface Membrane of Normal B Lymphocytes but Not B-Lineage Cell Lines",
"section_num": null
},
{
"section_content": "Because mouse B cells can express smBcl-2 during apoptosis (21), we ex- amined smBcl-2 expression on human B lymphocytes from B-CLL patients and normal subjects undergoing cell death.First, thawed cells, which would be expected to exhibit increased levels of apoptosis over fresh cells, were analyzed.Comparing these data to those from cells that had not been cryopreserved, the average percent smBcl-2 + cells in frozen/thawed samples was increased significantly compared with cells that had never been frozen and thawed (11.99 versus 23.41, respectively, P = 0.01; Figure 3A).Similar to fresh cells (see Figure 2), there was a significant difference in smBcl-2 levels between B-CLL cells and B cells from normal subjects (P = 0.03; see Figure 3A).However, there was no significant difference between M-CLL or U-CLL and normal controls in the thawed subset, possibly because of sample size (P = 0.056 and 0.16, respectively). Next, lymphoid populations from B-CLL patients were examined by flow cytometry, using forward scatter to divide the populations into smaller cells (R1), typically increased among dying/ dead cells, and larger (R2), more viable cells.smBcl-2 expression was markedly enriched in the smaller, dying/dead cell population than in larger, healthier cells.For patient 435, virtually all smBcl-2 was found on small cells, while, for patient 753, a lesser percentage, but still mostly the small cells, were smBcl-2 + (Figure 3B). To correlate smBcl-2 expression with the occurrence of apoptosis specifically, we analyzed freshly isolated B cells of both B-CLL and normal subjects by flow cytometry for smBcl-2 and cell viability, using either PI or Annexin V approaches.Consistent with other data sets, there was a significant difference in smBcl-2 + cells when comparing B-CLL and normal B cells (P = 0.02; Figure 4A, Supplemental Figure 2).The numbers of smBcl-2 + and Annexin V + cells were elevated in B-CLL compared with normal controls, suggesting that, as apoptosis proceeded, smBcl-2 expression increased. Finally, to directly examine the appearance of smBcl-2 during the apoptotic process, B cells were analyzed after inducing apoptosis by multiple methods: exposure to non-physiologic temperatures: 4°C (data not shown), 45°C (data not shown), and 60°C (Figure 4B, Supplemental Data Figure 3); or by exposure to camptothecin, a chemical that induces apoptosis in B-CLL cells (22) by affecting expression of Bcl-2 family members (23) (Figure 5A, Supplemental Data Figure 4).Figure 4B is representative of repeated experiments heating normal B-cells at 60°C for 3 min.Expos- ing normal B cells (n = 9) to these conditions always induced significant levels of apoptosis, but not always a significant increase in smBcl-2 expression.The corresponding t tests for cells heated 3 min (Supplemental Figure 3A,3B) are shown in Supplemental Data Figure 3D.Cells exposed to 60°C for 4 min induced over 98% Annexin V-binding cells with an increase in smBcl-2 of ~33%.Since the degree of smBcl-2 expression was not always proportional to the level of apoptosis induction at 3 or 4 min, it is difficult to draw a clear conclusion as to the relevance of smBcl-2.Of note, treating B-CLL cells with 10 µM camptothecin for 1 d significantly induced apoptosis in both B-CLL cells and normal aging B cells (P = 0.01; Figure 5A); however only B-CLL cells expressed significantly more smBcl-2 (P = 0.03; Figure 5A).Figure 5B illustrates an example of enhanced smBcl-2 expression by Annexin V high versus Annexin V low members of a B-CLL clone (note scale difference). ",
"section_name": "Relationship between smBcl-2 and Cell Viability in B-CLL and Normal Human B Cells",
"section_num": null
},
{
"section_content": "To further corroborate surface membrane expression of Bcl-2, confocal microscopy was performed (Figure 6).Similar to results obtained with flow cytometry (see Figure 2), heterogeneous patterns of smBcl-2 expression were observed, and some B-CLL clones contained more smBcl-2 + cells (Figure 6A) than others (Figure 6B,6C).smBcl-2 Detected in Other B-cell Malignancies Lacking t (14;18) To determine if smBcl-2 expression was unique to B-CLL, cells from other B-cell leukemias and lymphoproliferative disorders were analyzed.smBcl-2 was detected on B cells from patients with ALL (Figure 7A-D), HCL (Figure 7E), and MM (Figure 7F).Of note, smBcl-2 was found at high levels in virtually every ALL sample analyzed.Rows A-C illustrate data with unsorted samples, while rows D-F are data generated from cells sorted as described in Materials and Methods.The differences in ALL3 between rows A and D may represent cell debris generated by cell sorting.In addition, a difference was seen between two HCL cases studied (225 and 1317): levels of smBcl-2 + cells were similar (85.8% and 89.5%, respectively), although the subG1 apoptotic peak dominated for HCL225 but was only a minor population for HCL1317; thus, in this latter instance, more smBcl-2 was detected among viable cells. We also confirmed that smBcl-2 expression was enriched among dying cells from these other B-cell derived neoplasms, using simultaneous flow cytometry for smBcl-2 expression and PI analysis for DNA fragmentation (Figure 7D-F).These studies showed a clear correlation of smBcl-2 levels with the numbers of cells undergoing apoptosis in each disease. ",
"section_name": "Microscopic Examination of smBcl-2 Expression",
"section_num": null
},
{
"section_content": "In this study, we confirmed elevated Bcl-2 levels within B-CLL cells at the RNA and protein level.Microarray results from two different Affymetrix chips, U95 and U133, were verified by RT-QPCR and Western blot analyses (see Figure 1).We also uncovered the surprising presence of Bcl-2 on the surface of leukemic B cells, identified by flow cytometry (see Figure 2), and corroborated by confocal microscopy (see Figure 6).Although variable on a case-by-case basis, B-CLL cells had significantly increased levels of smBcl-2 (see Figure 2) on both fresh and frozen cells when compared with B cells from the blood of normal neonates and adult subjects.smBcl-2 expression has been reported in the murine thymocyte cell line, EL-4 (21), and its presence detected in the membrane fractions of cell lines and lymphomas with the t(14;18) translocation (24).Chen-Levy visualized low levels of smBcl-2 by phase contrast microscopy in a human follicular lymphoma cell line that carries the t(14;18) translocation.In a cell line retrovirally transfected to overexpress Bcl-2, the plasma membrane fraction contained Bcl-2 protein, while control cells did not (25), leading to the proposal that Bcl-2 localized to plasma membranes sup-pressed oxidative damage, as it does for mitochondrial membranes, and thereby had functional significance, possibly providing a survival advantage to these cells.Similarly, when Bcl-2 was observed in the membrane of mouse cells under-going apoptosis, it was proposed that Bcl-2, a known antioxidant (26)(27)(28), might maintain plasma membrane integrity as it does at the mitochondrial membrane. Although smBcl-2 was enhanced in dying cells, in several cases apparently healthy cells were smBcl-2 + , and, therefore, we cannot rule out a function for smBcl-2 in viable cells.Therefore, our finding of smBcl-2 occurring at lower levels on adult and cord blood B cell populations than on B-CLL cells suggests exaggeration of a normal function in leukemic clones.However, using surface markers to identify cellular health can be misleading at times.For example, smAnnexin A1 on HCL cells might lead to the false conclusion that these were compromised cells.In fact, Annexin A1 binding to the cell surface is seen with such consistency that it is now a marker for the disease (29).In this study, HCL cases exhibited a large percent of smBcl-2 + cells, as did previously cryopreserved ALL samples. In examining the functional relevance of smBcl-2 and its upregulation during apoptosis, it may be asked whether cells positive for smBcl-2 are utilizing the molecule in a functional manner or if the appearance of the protein in the plasma membrane is merely a reflection of altered lipid bilayer integrity.We currently do not have data supporting or excluding either possibility.Several rescue points have been identified during apoptosis (30,31).However, since smBcl-2 levels are increased in the small, probably dying, cell population identified by forward and side scatter (see Figure 3B) and by enhanced expression of Annexin V (see Figure 5B), but are not associated with DNA fragmentation defined by PI analysis (see Figure 4A), a mid-point during the apoptotic process is suggested as a targeted checkpoint by our studies.Takashina et al. have described rescue of HEK293 cells from apoptosis after activation of caspase 3 and appearance of membrane blebbing (32).This stage was associated with absent-to-low levels of Annexin V binding.Although the mech- anism responsible for the rescue of cells displaying membrane blebbing was not defined, upregulation of Bcl-2, along with cFLIP and IAP, did not occur.However, it should be mentioned that translocation of existing Bcl-2 to the plasma membrane was not considered.Although these findings are not consistent with prior findings indicating a checkpoint that precedes membrane blebbing (4,5), reversal of membrane blebbing also has been reported for pancreatic acinar cells over-stimulated by specific agonists (33).Although cancer cells presumably have many mechanisms to attempt to avoid cell death (for example, epigenetic silencing of DAPK1, a protein thought to assist in early apoptosis through promoter methylation that has been observed in almost all cases of sporadic B-CLL analyzed, (34), our data leave the possibility open that smBcl-2 may provide an additional advantage for the B-CLL clone during apoptosis.This may not be unreasonable considering the documentation that Bcl-2 and other family members become ER membrane-associated with stress (for example, excessive amounts of folded proteins [35], leading to survival activities such as autophagy [36]).In addition, the contribution of the Bcl-2 family to other activities not associated with apoptosis needs to be considered, that is, involvement in the cell cycle (37), repair of damaged DNA (37,38), and metabolism of glucose (39). In summary, the presence of Bcl-2 on the surface membrane of B-CLL cells and B cells of certain other lymphoproliferative disorders is a surprising finding, although, at this juncture, it is not clear if smBcl-2 expression is a passive consequence of the apoptotic process or an active attempt to abort cell death by stabilizing the surface membrane.Further investigations of the functional relevance of this finding are warranted. ",
"section_name": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "The authors thank Herb Borerro, Tom McCloskey, Stella Stefanova, and Amanda Chan of The Feinstein Institute, Manhasset, New York, United States of America, and Carolyn Cheney of Ohio State University, Columbus, Ohio, United States of America, for their help with flow cytometric and confocal microscopic analyses.The Bcl-2-overexpressing variant of 697, 697-Bcl-2, and its empty-vector control, 697-Neo22, were the kind gift of Drs.Shinichi Kitada and John Reed of the Burnham Institute for Medical Research, La Jolla, California, United States of America.We also are grateful to N Muthusamy, Ohio State University, for assistance with the 697 cell experiments, and to Gloria Telusma, Rahena Chowdhury, and Somara Rafiq for assistance with B cell isolation.Finally, we acknowledge the insights of Drs.Patricia Mongini, Michael Grever, and John Byrd in preparation of the manuscript. These studies were supported in part by RO1 grant CA 87956 from the NIH National Cancer Institute, M01 General Clinical Research Center Grant (RR018535) from the NIH National Center for Research Resources, and fellowship grants from the Lauri Strauss Leukemia Foundation.The Karches Foundation, Peter Jay Sharp Foundation, Prince Family Foundation, Marks Foundation, the Jean Walton Fund for Lymphoma and Myeloma Research, and the Joseph Eletto Leukemia Research Fund also provided support for these studies. ",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
}
] |
10.1038/s42003-023-04921-z
|
Functional impact and molecular binding modes of drugs that target the PI3K isoform p110δ
|
<jats:title>Abstract</jats:title><jats:p>Targeting the PI3K isoform p110δ against B cell malignancies is at the mainstay of PI3K inhibitor (PI3Ki) development. Therefore, we generated isogenic cell lines, which express wild type or mutant p110δ, for assessing the potency, isoform-selectivity and molecular interactions of various PI3Ki chemotypes. The affinity pocket mutation I777M maintains p110δ activity in the presence of idelalisib, as indicated by intracellular AKT phosphorylation, and rescues cell functions such as p110δ-dependent cell viability. Resistance owing to this substitution consistently affects the potency of p110δ-selective in contrast to most multi-targeted PI3Ki, thus distinguishing usually propeller-shaped and typically flat molecules. Accordingly, molecular dynamics simulations indicate that the I777M substitution disturbs conformational flexibility in the specificity or affinity pockets of p110δ that is necessary for binding idelalisib or ZSTK474, but not copanlisib. In summary, cell-based and molecular exploration provide comparative characterization of currently developed PI3Ki and structural insights for future PI3Ki design.</jats:p>
|
[
{
"section_content": "T he treatment of B lymphoid malignancies has been revolutionized by targeting kinases involved in B cell receptor (BCR) signaling, which include the phosphoinositide-3kinase (PI3K) isoform δ 1 .Thus, disrupting the activity of p110δ for these indications resulted in the first clinical approvals of PI3K inhibitors (PI3Ki) 2 .Idelalisib, the first clinically applied PI3Ki, is an effective treatment of chronic lymphocytic leukemia (CLL) 3 , but limited by immune-related adverse events and resistance 4,5 .Among targeted agents, PI3Ki therefore currently plays a less prominent role in the treatment of CLL compared to BTK inhibitors and BH3 mimetics 6 . PI3Ks catalyze the phosphorylation of the membrane constituent phosphoinositide-(4,5)-biphosphate (PIP2) to yield phosphoinositide- (3,4,5)-triphosphate (PIP3) 2 .By interaction of their pleckstrin-homology (PH) domains with PIP3, downstream mediators are recruited to the plasma membrane, where they are activated.Among these, the serine threonine kinase AKT serves as an important signaling hub and AKT phosphorylation is commonly used to monitor PI3K activity in cells.The four class I PI3K catalytic subunit isoforms have distinct though overlapping roles in pivotal physiological cell functions as well as in oncogenic signaling 7 .In this regard, the high frequency of activating point mutations in p110α, e.g., H1047R, in solid tumors points to an important targetable oncogenic potential 8,9 .In contrast, the similar E1021K substitution in p110δ occurs as a rare germline mutation that causes activated PI3K delta syndrome (APDS) 10 .Nevertheless, we employed this mutation to generate a p110δdependent mechanistic cellular model for PI3Ki assessment.The catalytic PI3K subunit p110δ further differs from p110α by preferential expression in hematopoietic tissues and physiological roles in antigen-receptor signaling and immune responses 11 .Since p110δ is overexpressed and constitutively activated by external stimuli, including the BCR, the survival of CLL cells, as well as their cytokine secretion and chemotaxis, can be targeted by idelalisib 12,13 .While genetic inactivation and isoform-selective pharmacologic targeting have largely explained the functional involvement of p110δ, these findings can be corroborated and refined by a chemical genetic approach, in which idelalisibinhibited cell functions are rescued by a resistance mutation, such as the newly identified I777M substitution. Among pharmacological PI3Ki, multi-targeted substances can be distinguished from p110δ-selective molecules that interact with a cleft between W760 and M752 of p110δ 14 .Consequently, mutation of M752 in p110δ leads to resistance against propellershaped p110δ-selective PI3Ki, whereas binding of flat multitargeted PI3Ki remains unaffected.Comparisons of p110δ apo and PI3Ki-bound structures demonstrated an induced fit of propeller-shaped inhibitor molecules into the mentioned cleft, which is designated as a selectivity or specificity pocket 15 .Apart from this mechanism, the isoform selectivity of PI3Ki is determined by the inner core of the binding site, including nonconserved regions and conserved affinity pocket residues not directly contacting ATP 16,17 .To create drug-resistant p110δ, we introduced the I777M substitution that is analogous to the I800M mutation in p110α, which causes resistance to a few tested multitargeted PI3Ki 18 .In some instances, this substitution close to the catalytic K779 was compared to the I825V mutation that affects the so-called gatekeeper residue, which controls inhibitor access to a pre-existing cavity within the ATP-binding pocket of protein and lipid kinases 19 .The I777M substitution allows the combined structural and cell-based examination of its impact on the binding of diverse PI3Ki as a contribution to ligand design. To elucidate the molecular pharmacology of p110δ, we used isogenic cell lines expressing variants of this PI3K isoform.As a starting point, the activating C-terminal E1021K mutation enabled the development of a sensitive, isoform-selective mechanistic cellular model that we subsequently used for comparative high-throughput PI3Ki assessment.In addition, we employed the affinity pocket mutation I777M in p110δ, which caused resistance to idelalisib and thus provided the opportunity to explore the engagement of p110δ in BCR-dependent and oncogenic cell functions.The I777M mutation also served as a probe for p110δ interactions with diverse PI3Ki chemotypes in the aforementioned cell line-based assay system.The results of this assessment were related to structural investigation using molecular dynamics (MD) simulations. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Oncogenic properties of p110δ.As a prerequisite for PI3Ki characterization via cell viability, we examined the oncogenic potential of wildtype (wt) and mutant p110δ in the untransformed murine pro-B cell line BaF3, which requires interleukin 3 (IL-3) for growth under normal cell culture conditions, but can be rendered IL-3-independent by expression of different tyrosine kinases 20 or p110α-H1047R 21 .For this purpose, BaF3 cells were retrovirally transduced to express variants of p110α and p110δ at approximately equal levels in BaF3 cells (Supplementary Fig. 1a).Under IL-3 withdrawal, the viability of these constructs revealed the degree of factor-independence owing to overexpression and point mutations (Supplementary Fig. 1b).Expression of p110δ or p110α increased the percentages of IL-3-independent cells from 6 to ~20%.The E1021K or H1047R mutations augmented IL-3independence to 30 or 50%, respectively, and further to above 50% in combination with binding pocket mutations in both isoforms.While in p110α, the main increase was due to the H1047R mutation, p110δ on its own showed slightly higher transformation capacity than p110α and only a slight increment with the E1021K mutation.To further explore the oncogenic potential of PI3K isoform variants, we used a different oncogenicity assay, namely anchorage-independent growth of murine fibroblasts 22 .In NIH3T3 cells the formation of colonies with an area of more than 1200 µm 2 in soft agar was increased approximately threefold by each, p110δ overexpression and the I777M mutation, which together reached only half of the effect of p110α-H1047R (Supplementary Fig. 1c). To apply p110 isoform-dependent BaF3 cells for PI3Ki assessment, we modified the assay procedure to include a preincubation period of several days in the absence of IL-3.This combined selection for vector-encoded antibiotic resistance and factor-independent growth led to higher percentages of IL-3independent viability and to a reduced difference between isogenic BaF3 cells with only E1021K or with additional I777M mutation (Fig. 1a).Subsequently, we investigated the concentration-dependent inhibition by idelalisib of the IL-3independent growth of isogenic BaF3 cells expressing wt and mutants of p110δ (Fig. 1b).BaF3 cells expressing p110δ-wt showed higher sensitivity to idelalisib than parental BaF3 cells grown in IL-3-complemented medium.The activating E1021K mutation caused a further increase in sensitivity to idelalisib, resulting in a cellular half maximal inhibitory concentration (IC 50 ) in the order of 100 nM.To prove that we had generated a PI3K isoform-selective system for PI3Ki assessment, we used transduced BaF3 cells that express p110δ-E1021K or p110α-H1047R for determining the cellular sensitivity of three additional PI3Ki with known differences in efficacy and isoform selectivity (Fig. 1c).While duvelisib showed a strong preference for inhibiting p110δ, alpelisib affected preferentially the growth of p110α-dependent BaF3 cells and copanlisib led to strong and apparently equal inhibition of the cells transformed by both PI3K isoforms.These examples show that monitoring the cell viability of PI3K isoform-dependent BaF3 cells can serve as a tool for comparatively assessing the potency and isoform selectivity of PI3Ki in a cell-based system that is amenable to high-throughput measurements. PI3Ki profiling by p110δ-dependent BaF3 cells.To demonstrate their usefulness for PI3Ki assessment, we employed BaF3 cells expressing p110δ-E1021K for characterizing a larger set of structurally diverse PI3Ki.Owing to the common distinction of isoform-selective and multi-targeted PI3Ki 14 , we classified these PI3Ki according to published biochemical isoform selectivity (Supplementary Fig. 2 and Supplementary Table 1).Multitargeted PI3Ki with less than 14-fold selectivity to one than the other isoforms were subdivided into pan-class PI3Ki and dual PI3K/mTORi according to the presence or absence of potency against mTOR.The sensitivity of isogenic p110 isoformdependent BaF3 cells to a collection of PI3Ki at concentrations from 2 nM to 10 µM was assessed by viability assays on 384-well plates. Since isogenic BaF3 cells distinguished the isoform selectivity of example PI3Ki (Fig. 1c), we determined the ratios of cellular IC 50 values for p110δ and p110α of 27 PI3Ki (Fig. 2a).In a ranking according to isoform selectivity, p110δ-and p110αselective PI3Ki flanked a range of multi-targeted PI3Ki, among which ZSTK474 exhibited high selectivity for p110δ.At the extremes, duvelisib and serabelisib showed 2660-fold or 3.8-fold cellular selectivity for p110δ or p110α, respectively.Overall, a median of 3.6-fold higher cellular than biochemical selectivity for p110δ corresponded to enhanced sensitivity of p110δ-dependent BaF3 cells to this isoform.The present assessment of a PI3Ki library proved the capability of isogenic BaF3 cells to resolve the isoform selectivity of PI3Ki and to translate biochemical to cellular PI3Ki isoform selectivity. Drug sensitivity testing using isogenic BaF3 cells showed a wide range of responses among the examined PI3Ki with pronounced differences between p110δ-and p110α-dependent BaF3 cells (Fig. 2b and Supplementary Table 2).Regarding p110δ as a relevant drug target in hematological malignancies, biochemical and cellular PI3Ki potencies of the PI3Ki in the present collection were largely in agreement (Supplementary Fig. 3a,b).A ranking of the examined PI3Ki by their cytotoxicity to BaF3 cells expressing p110δ-E1021K, indicated several substances with higher potencies than the clinically used and prototypic p110δselective PI3Ki idelalisib, namely nine multi-targeted and the p110δ-selective PI3Ki duvelisib and AMG319.In summary, BaF3 cells expressing p110δ-E1021K sensitively indicate differences in cellular PI3Ki potencies that generally relate well with reported biochemical potencies. In addition to engineered BaF3 cells, we used the activated B cell-like (ABC) diffuse large B cell lymphoma (DLBCL) cell lines TMD8 and HBL-1 as tools for PI3Ki characterization (Fig. 2b).TMD8 cells responded considerably less to p110δ-selective PI3Ki than p110δ-dependent BaF3 cells, but their sensitivity to multitargeted and p110α-selective PI3Ki followed a similar ranking (Supplementary Fig. 3c,d).HBL-1 cells showed at least 20-fold Fig. 1 IL-3-independent growth and PI3Ki sensitivity of isogenic BaF3 cells.BaF3 cells were retrovirally transduced to stably express the wild type or the indicated mutants of p110α and p110δ.After pre-incubation in media without IL-3 for several days, cell viability after four days in the absence of IL-3 was determined by bioluminescence measurements of ATP amounts with the CTG assay on 384-well plates.The viability of different isogenic BaF3 cells in the absence of IL-3 is expressed as a percentage of IL-3-complemented parental cells.P values for differences to parental BaF3 cells and indicated comparisons were determined by two-sided, unpaired T-tests.a IL-3-dependent growth of BaF3 cells expressing p110α and p110δ with C-terminal activating mutations alone or combined with additional binding pocket mutations was recorded.Per cell type, the box plots represent twelve measurements from two independent experiments.b The concentration-dependent cytotoxicity of idelalisib for isogenic BaF3 cells was determined under IL-3 deprivation for BaF3 cells expressing p110δ-wt or p110δ-E1021K and for parental BaF3 cells in the presence of IL-3.Means and single values of a representative quadruplicate measurement among four repetitions are shown.c The concentration-dependent responses to the PI3Ki duvelisib, alpelisib, and copanlisib were recorded in BaF3 cells expressing p110α-H1047R or p110δ-E1021K.Representative experiments were taken from three or four repetitions, respectively.Error bars indicate standard deviations among quadruplicate measurements.Fig. 2 Cellular isoform selectivity and potency of structurally diverse PI3Ki.The efficacy of a collection of PI3Ki was assessed in concentrationdependent viability assays with BaF3 cells expressing p110δ-E1021K or p110α-H1047K.Presented data are restricted to substances that show lower cellular IC 50 values than 10 µM against at least one of the investigated p110 isoforms.a For isoform selectivity profiling, PI3Ki were ranked according to the ratios of cellular IC 50 ratios of p110α/p110δ and compared with the corresponding ratios from published biochemical IC 50 values.Cellular and biochemical IC 50 ratios are presented by empty and filled bars, respectively.b The investigated PI3Ki were ranked according to their potency against BaF3 cells expressing p110δ-E1021K and compared with the efficacy of the same PI3Ki against p110α-H1047R and the DLBCL cell lines TMD8 and HBL-1.Compound names of pan-class I PI3Ki are printed in black.Blue, green, and orange print indicates PI3Ki selectivity for p110δ, p110α, and p110γ, respectively.lower sensitivity than TMD8 cells to all examined p110δ-selective PI3Ki and a few multi-targeted PI3Ki.Among control substances, TMD8 cells showed lower cellular potencies to inhibitors of BTK than p110δ-dependent BaF3 cells (Supplementary Fig. 4a,b).In summary, p110 isoform-dependent BaF3 cells combine sensitive isoform-selective PI3Ki assessment and high biological relevance. Idelalisib resistance caused by a binding pocket mutation.We investigated the I777M mutation in p110δ, because the analogous mutation in p110α mediated resistance to some PI3Ki 18 .For this purpose, we transduced malignant B cell lines for stable expression of mutant and wt p110δ at comparable levels and monitored AKT phosphorylation as an indirect measure of PI3K activity.In Ramos cells, which can be activated by clustering the BCR with anti-IgM, AKT phosphorylation at S473 more than doubled after treatment with anti-IgM (Supplementary Fig. 5a).Transgene expression and pAKT levels were also followed in BaF3 cells with and without IL-3 stimulation (Supplementary Fig. 5b).Compared to wt, AKT phosphorylation was increased by the I777M mutation in both cell lines, while the gatekeeper mutation I825V had the opposite effect. Concentration-dependent immune blot analysis of anti-IgMstimulated Ramos cells indicated that the I777M mutation maintained AKT phosphorylation in the presence of idelalisib (Fig. 3a).Compared to wt, p110δ-I777M significantly rescued AKT phosphorylation at 10 and 100 nM idelalisib.A similar effect of the I777M mutation on pAKT levels was observed in BaF3 cells (Supplementary Fig. 6b).In contrast, the gatekeeper mutation I825V, which strongly reduced AKT phosphorylation in activated Ramos and BaF3 cells, enhanced the sensitization to idelalisib owing to p110δ overexpression (Supplementary Fig. 6a,c).In NIH3T3 cells, which are essentially devoid of endogenous p110δ expression, forced expression of p110δ-wt led to increased sensitivity to idelalisib (Supplementary Fig. 7a).This sensitization to idelalisib owing to overexpression of p110δ was less pronounced in Ramos and BaF3 cells with increasing endogenous p110δ expression (Supplementary Fig. 7b,c).To extend concentration-dependent signaling analyses to additional PI3Ki, anti-IgM-stimulated Ramos cells were also examined by phospho-specific flow cytometry (Fig. 3b).Idelalisib, duvelisib, and copanlisib differentially inhibited the AKT phosphorylation in Ramos cells expressing p110δ-wt.Compared to wt, Ramos cells expressing p110δ-I777M were approximately 100 times less sensitive to idelalisib and duvelisib and equally sensitive to copanlisib.Thus, according to AKT phosphorylation, the I777M mutation in p110δ mediated strong resistance to idelalisib. Rescue of cell functions in the presence of idelalisib.Maintained AKT phosphorylation in the presence of idelalisib owing to the I777M mutation in p110δ enabled chemical genetic evaluation of the functions of malignant B cells.For this purpose, we examined the concentration-dependent inhibition by idelalisib of cytokine secretion in Ramos cells with activated BCR signaling (Fig. 4a).Overexpression of p110δ-wt augmented the anti-IgMinduced secretion of CCL3, but not of CCL4 and TNFα compared to the vector control (Supplementary Fig. 8a).The I777M mutation further increased the secretion of CCL3 but not of CCL4 and TNFα compared to wt in a similar manner, and, importantly, maintained the secretion of all three cytokines in the presence of idelalisib (Fig. 4a).In contrast to metabolic activity (Supplementary Fig. 8b), the I777M mutation reconstituted cytokine secretion of Ramos cells in the presence of idelalisib. In addition, we investigated the transwell migration to CXCL12 of BaF3 cells expressing wt or mutant p110δ.Chemotaxis to CXCL12 increased the number of migrating cells approximately one-thousand-fold compared to spontaneous migration (Fig. 4b).BaF3 cells expressing p110δ-I777M showed approximately threefold higher spontaneous migration than wt or vector controls.Compared to wt, p110δ-I777M significantly maintained the chemotaxis of BaF3 cells to CXCL12 in the presence of 1 µM idelalisib (Fig. 4c).Rescue of cytokine secretion and cell migration in the presence of idelalisib by the I777M mutation documented p110δ engagement in B cell functions involved in the microenvironmental dialog. Mutation-mediated resistance to diverse PI3Ki.For further characterization of the I777M substitution, we took advantage of our newly developed p110δ-selective cell line model harboring the activating E1021K mutation.Indeed, these p110δ-dependent BaF3 cells indicated viability as a further crucial cell function that can be reconstituted by the I777M mutation for the evaluation of PI3Ki resistance (Fig. 5).The I777M mutation significantly rescued the viability of isogenic BaF3 cells at several concentrations of idelalisib, duvelisib, umbralisib, and ZSTK474, but not copanlisib (Fig. 5a,b).In a larger set of PI3Ki arranged according to decreasing cellular PI3Ki potency, we recorded changes of the cellular IC 50 in p110δ-dependent BaF3 owing to the I777M mutation (Fig. 5c).While this substitution caused prominent resistance mainly to p110δ-selective PI3Ki, the analogous I800M mutation in p110α showed strong resistance to most multitargeted PI3Ki, particularly the three most potent ones (Supplementary Fig. 9a).In addition, we ranked PI3Ki according to the strength of mutation-mediated resistance expressed as IC 50 ratios (Supplementary Fig. 9b, c and Supplementary Table 3).The maximal observed resistance owing to p110δ-I777M or p110α-I800M was 25-or 480-fold with duvelisib or copanlisib, respectively.Remarkably, p110δ-I777M led to sensitization to some PI3Ki, predominantly those with additional activity against mTOR.The I777M mutation in p110δ-dependent BaF3 cells did not result in apparent resistance to other drugs than PI3Ki (Supplementary Fig. 4c,d).Individual PI3Ki often showed opposite positions in the resistance rankings determined for p110δ and p110α.Apart from p110δ-selective PI3Ki, the I777M mutation in p110δ caused strong resistance to ZSTK474, but none to copanlisib.Together with the availability of structural data (Supplementary Table 4), these observations defined the selection of substances for the analysis of binding pocket interactions by molecular dynamics simulations, namely of idelalisib as representative of p110δ-selective PI3Ki in comparison with the pan-class I PI3Ki ZSTK474 and copanlisib. Structural and dynamical changes in p110δ caused by the I777M substitution.To explain our pharmacological observations at the molecular level, we investigated the structural basis of the interaction of PI3Ki with p110δ.For this purpose, we performed MD simulations of structural models of human p110δ-wt and -I777M with and without small molecule inhibitors bound (Supplementary Table 5).According to backbone fluctuations, the I777M substitution increased the overall flexibility of the p110δ apo-structure, particularly in the P-, catalytic, and activationloops and the hinge region (Supplementary Fig. 10a). Comparisons of apo and idelalisib-bound p110δ-wt (Supplementary Movies 1, 2) showed an induced fit of the quinazoline moiety of idelalisib into the specificity pocket between M752 and W760, which did not form in p110δ-I777M (Fig. 6a).Idelalisib binding to p110δ-wt was reinforced by a very stable hydrogen bond between V828-NH in the hinge region and a nitrogen from the purine moiety of idelalisib, which was exhibited all over the trajectory (Supplementary Fig. 10b,d).In addition, there were water-mediated hydrogen bonds between idelalisib and residues M900 and I910 of the hydrophobic region II adjacent to the activation loop of p110δ-wt (Supplementary Fig. 10b,d).In p110δ-I777M, however, a conformational rearrangement of the ATP-binding pocket occurred, that resulted in a steric clash of the M777 side chain with idelalisib as positioned in p110δ-wt (Fig. 6b,c).Therefore, idelalisib adopted an altered position and came close to different residues in p110δ-I777M than, in p110δwt, namely Y813 as well as S831 and T833 of the hinge region (Supplementary Fig. 10c).The altered conformation of p110δ-I777M resulted in the joint shift of M752 and W760 side chains and thus prevented the opening of the specificity pocket, which resulted in the displacement of idelalisib (Fig. 6a and Supplementary Movie 2).Accordingly, the volume of the idelalisib binding pocket was reduced from 643 Å 3 in p110δ-wt to 357 Å 3 in p110δ-I777M (Fig. 6d,e).Disruption of the specificity pocket in p110δ-I777M was reflected by significant changes compared to p110δ-wt in additional structural features, e.g., distances, angles, and binding pocket surface area (Supplementary Fig. 10d-f).Overall, MD simulations provided clear-cut evidence that the I777M substitution led to a conformational change that caused idelalisib resistance by preventing the formation of the specificity pocket between M752 and W760. Despite its classification as pan-class I PI3Ki according to isoform selectivity, ZSTK474 was subject to I777M-mediated resistance in cell-based assays.MD simulations of ZSTK474 binding to p110δ-wt suggested that the benzimidazole group was accommodated in a pocket formed by residues I777 and K779 that was smaller in the absence of the inhibitor (Fig. 7a and Supplementary Movie 3).One of the morpholino groups was positioned close to the hinge region (residues I825-V828) with the kinase N-and C-lobes approaching each other.One fluor atom of the difluoromethyl group pointed towards K779 the other lined up with one of the two morpholino groups, rotated by 90°, both projecting out of the pocket and highlighting a non-planar ZSTK474 geometry (Fig. 7a and Supplementary Movie 4).A hydrogen bond was established between the nitrogen of the benzimidazole of ZSTK474 and the amino group of K779 (Supplementary Fig. 11c,d).The I777M substitution, however, prevented ZSTK474 from entering into the pocket, since the opening between I777 and K779 was no longer available (Fig. 7b).Though smaller than the specificity pocket induced upon idelalisib binding, the ZSTK474 binding pocket volume was larger in wt than mutant p110δ.Compared to wt, the difluoromethyl group changed the orientation facing upwards to M752 and P758 (Supplementary Fig. 11a).ZSTK474 moved closer to K779 and D787 with one morpholino group approaching T833.Several features in the molecular geometry of ZSTK474 binding indicated significant differences between p110δ-wt and -I777M (Supplementary Fig. 11b).For ZSTK474, the I777M substitution led to resistance due to conformational changes that mainly involved residues of the affinity pocket. To find out why the I777M mutation did not mediate resistance to copanlisib in cellular assays in contrast to idelalisib and ZSTK474, we performed MD simulations of this PI3Ki molecule bound to p110δ-I777M.The position of copanlisib extended from residues capable of forming a specificity pocket via the adenine and affinity pocket towards N898 and D911 at the mouth of the ATP-binding pocket and close to the activation loop (Fig. 7c and Supplementary Movie 5).Compared to idelalisib and ZSTK474, copanlisib exhibited a less rotatable and flexible geometry.Hence, even though M777 was within 3 Å to all three PI3Ki, it obstructed the binding of idelalisib and ZSTK474, but not copanlisib. In addition to M777, residues M752, V827, T833, M900, and I910 were common contact residues of the three inhibitors.ZSTK474 exclusively contacted residues K779, D787, E826, and T833 of the extended affinity pocket and idelalisib residue S831.Taken together, our in silico findings show, how the I777M substitution precluded the binding of idelalisib and ZSTK474 to p110δ inducing a conformational rearrangement of the binding site, while copanlisib binding was not affected by the I777M mutation. ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Using C-terminal activating mutations of p110, we developed a cell-based high-throughput system for the assessment of PI3Ki isoform selectivity and potency.Moreover, we exploited the affinity pocket mutation I777M to evaluate p110δ engagement in BCR-dependent and oncogenic cell functions and to provide an experimental basis for linking cell-based assessment with structural models of PI3Ki interactions (Fig. 8). To generate a system for cell-based, p110δ-selective PI3Ki assessment, we took advantage of the transformation of BaF3 cells to factor-independence owing to the E1021K mutation in p110δ 10 , similarly as previously performed with p110α-H1047R in a different cell line 18 .The IL-3 independence gained by binding.The difluoromethyl group of ZSTK474 fits between residues I777 and K779 (gray mesh) of p110δ-wt, but not between M777 and K779 of mutant p110δ (red mesh).The color of the mesh around M777 indicates sterically hindered (red) or undisturbed (gray) inhibitor binding.Moreover, the I777M substitution reduces the size of the binding pocket (red), from which the ZSTK474 molecule is expelled.c Different molecular interaction of idelalisib, ZSTK474 and copanlisib with p110δ-I777M.In the bar plots on the top, binding pocket residues are arranged from N to C terminus and assigned to regions of the specificity pocket (yellow), adenine and affinity pocket (blue), excluding an extended hinge region (gray), as well as catalytic and activation loop (purple).The height of each bar represents the total number of frames where the corresponding residue was found within a 3 Å distance to the inhibitors out of 1000 from each simulation.Only residues found in more than 75% of the frames are shown.The residues displayed in the bottom panel correspond to those shown in the top panel and are located within the binding pocket of individual inhibitors.Each residue is assigned a region-specific color according to the bar plots.The inhibitors are represented as balls and sticks and the residues as lines.Of note, the enzyme ligand structures show disturbed interactions with idelalisib and ZSTK474 in contrast to copanlisib binding, that is not subject to mutation-mediated resistance.For these three PI3Ki, the residues within a 3 Å distance illustrate interaction with different binding pocket regions. transduction of BaF3 cells with p110δ and p110α controls was in concordance with the reported transformation potential of these molecules for fibroblasts 23,24 .The E1021K mutation in p110δ, similar to p110α-H1047R 21 , oncogene fusions of tyrosine kinases 25 or myristoylation of AKT 26 secured high inhibitor sensitivity of the IL-3-independent viability of BaF3 cells.The ratios of IC 50 values obtained for the examined PI3Ki with isogenic p110δ-and p110α-dependent BaF3 cells spanned four orders of magnitude, which surpassed the resolution of isoform selectivity observed in an analysis of isoform-selective AKT inhibitors in a similar BaF3 cell-based system 26 .In contrast, the few cellular mechanistic models hitherto applied for PI3Ki assessment relied on the detection of AKT phosphorylation rather than cell viability 27,28 .For three PI3Ki in our study, the viability of p110δ-dependent BaF3 cells (Fig. 4a) showed similar concentration-response relationships as pAKT levels in Ramos cells with activated BCR (Fig. 3b) at approximately 10-fold lower sensitivity.Compared to a set of diverse cell lines with predominantly activated PI3K class I isoforms 27 , p110δ-and p110αdependent BaF3 cells provided a coherent assay system that potentially can be extended by BaF3 cells expressing mutationally activated p110β and p110γ.Another mechanistic cellular system for PI3Ki assessment, namely fibroblasts expressing myristoylated class I PI3K isoforms 28 , uses more rigid PI3K activation than C-terminal activating mutations and a cell context that is less related to B cell malignancies than our newly established system for PI3Ki profiling via straightforward viability measurement.With these advantages compared to existing systems, our newly developed mechanistic model not only allowed the determination of isoform-selective cellular PI3Ki potencies, but also served to evaluate the effects of the affinity pocket mutation I777M on p110δ interactions with PI3Ki. We introduced this mutation close to the catalytic K779 in p110δ to explore whether it renders the enzyme opaque to pharmacological inhibitors.Its characterization in Ramos cells revealed that, indeed the I777M substitution mediated resistance to idelalisib and, in addition, unexpectedly led to increased pAKT levels corresponding to a gain of function (Fig. 3a, b and Supplementary Fig. 5), whereas the analogous mutation in p110α moderately reduced PI3K activity 18 .Co-occurrence in p110δ of I777M and E1021K point mutations resulted in enhanced transforming potential, which is reminiscent of the combined effect of two kinase domain mutations in BTK 29 .The I777M substitution reconstituted pAKT levels at ~100-fold increased idelalisib concentrations, which is similar to the dasatinib resistance mediated by the gatekeeper mutations in the BCRassociated kinases BTK or LYN and higher than the ibrutinib resistance owing to the C481S mutation in BTK that prevents irreversible inhibitor binding 30 .Although the I777M mutation at the affinity pocket of p110δ functionally impedes the efficacy of idelalisib and thus essentially could presage acquired resistance to this drug, whole exome sequencing (WES) did not detect recurrent mutations in p110δ in samples from patients with disease progression on idelalisib 5 .This is corroborated by the fact that specifically in the case of PI3Ki, it is notoriously difficult to generate drug-resistant cell lines through in vitro exposure over long periods of time 9 , as well as by WES analysis of idelalisibresistant TMD8 cells generated in this manner 31 and in an in vivo model using serial tumor transfer and treatment 32 .In contrast to clinically acquired mutations in the tyrosine kinases Abl 33 and BTK 34 , resistance to idelalisib is mostly based on compensatory mechanisms, such as PTEN loss and up-regulation of PI3Kγ expression 31 , upregulated IL-6 and PDGFR signaling 35 , activation of IGF1R 32 or mutations that activate the MAPK signaling cascade 36 .In summary, the I777M functionally mediates resistance to idelalisib, leaving the intriguing question, of why this binding pocket mutation is not selected for in vivo, in contrast to similar mutations in tyrosine kinases. The cell-based assay systems used in this study relied on the expression of p110δ variants in addition to endogenous wt p110δ.Since endogenous p110δ levels increased from NIH3T3 to Ramos and BaF3 cells, overexpression of p110δ increased in this order and led to correspondingly augmented sensitivity to idelalisib (Supplementary Fig. 7a-c).This sensitization by p110δ overexpression can be attributed to p110δ-selective PTEN activation by still unknown mechanisms, conceivably protein kinase activity of p110δ or compartmentalized signaling 37,38 .This effect can lead to dampened resistance to dual inhibitors of PI3K and mTOR and even amount to overall sensitization due to the concomitant blockade of an independent target further downstream in the same pathway (Supplementary Fig. 7d).Moreover, the contribution of p110δ overexpression to oncogenic transformation by far exceeded that of p110α, presumably owing to the pro-B cell nature of the parental BaF3 cells.Both, sensitization to inhibition of p110δ and biased p110δ dependence and, may have contributed to shifting the observed cellular isoform selectivity of PI3Ki towards p110δ compared to the biochemically determined values.Background expression of p110δ-wt did not disturb the Fig. 8 The PI3K isoform δ as a drug target.An activating mutation (blue) in p110δ enables comparisons of the cellular potencies of diverse PI3Ki in viability assays.The affinity pocket mutation I777M (red) consistently mediates resistance to p110δ-selective PI3Ki, but only occasionally to pan-class I PI3Ki.In accordance with cell-based assays, MD simulations indicate resistance owing to the I777M substitution for idelalisib and ZSTK474, which project from the plain occupied by ATP, but not for the flat copanlisib molecule.MD simulations also show how the I777M substitution leads to conformational changes that can affect PI3Ki interactions with p110δ.Three-dimensional poses of PI3Ki interacting with p110δ-I777M were taken from the equally oriented structures in Fig. 7c. analysis of resistance, since PI3Ki treatment exerted selection pressure against it.Taking the mentioned limitations into account, the generated cell-based systems accurately indicated isoform-selective PI3Ki effects and resistance in a cell context. The present PI3Ki assessment focused on the inhibition of p110δ as the predominant isoform in B cell malignancies and used a PI3Ki collection that included the five PI3Ki currently approved for clinical use and the irreversible PI3Ki sonolisib.For this library of structurally diverse PI3Ki, our newly established p110δ-dependent cell-based system showed lower potencies among p110δ-selective than multi-targeted PI3Ki in accordance with earlier observations 14 .Apart from p110δ-selective PI3Ki, the ranking of PI3Ki efficacy against p110δ-dependent BaF3 cells resembled that against DLBCL cell lines.TMD8 and HBL-1 cells with low and high p110α expression, respectively, corroborated the advantage of additionally targeting p110α, e.g., by copanlisib and AZD8835 39,40 , which also was observed in malignant B cells from mantle cell lymphoma and CLL 41,42 .Overall, we created a resource of PI3Ki characterization that connected isoformselective and disease-relevant cellular PI3Ki potencies for comparisons with patient-specific or other pre-clinical PI3Ki assessment, e.g., in CLL or DBLCL cells 43,44 , and for the extension to the analysis of PI3Ki resistance. Although apparently not involved in clinical idelalisib resistance, the I777M substitution provides an exquisite tool for dissecting the roles of PI3K isoforms in a cell context.The dependence of the cytokine secretion by Ramos cells on stimulation of the BCR and increased CCL3 production owing to p110δ overexpression attest to a prominent role of this PI3K isoform as a BCR-associated kinase.In particular, the concentrationdependent response to idelalisib of Ramos cells expressing wt and resistance mutant of p110δ was more directly translated from pAKT levels to CCL3 secretion than the corresponding responses to ibrutinib and dasatinib to activation of BTK and Src family kinases that had been investigated in analogous systems expressing wt and mutant BTK and LYN 30 , indicating closer linkage of BCR-activated CCL3 secretion with the PI3K/AKT than the MEK/ERK or PLCγ/PKCβ signaling axis.Enhanced oncogenicity and rescue of directed transwell migration in the presence of idelalisib by the I777M mutation also proved engagement of p110δ in basal and CXCL12-dependent mobility of BaF3 cells, respectively.According to chemical genetic evaluation, p110δ was engaged in cell functions with importance for the microenvironmental dialog, e.g., cytokine secretion and chemotaxis, but also in p110δ-driven cell viability.The newly developed toolset for the functional evaluation of PI3K isoforms can be applied to models representing tumor cells as well as cell types of the tumor micro-environment, where BCR-associated kinases also play relevant roles 45 . Our cell-based PI3Ki assessment indicated that resistance due to the I777M mutation in p110δ mainly concerned p110δ-selective PI3Ki.For their prototypic representative, idelalisib, MD simulations allowed to directly follow induction of the specificity pocket upon ligand binding, as previously concluded from X-ray structures of co-crystals 15,46 , and showed that the I777M mutation disturbed its formation via conformational changes that affected the position of M752.The most prominent structural change owing to the I777M substitution observed in the interaction of p110δ and idelalisib occurred distantly from the mutation site at this residue that is known to be involved in the binding of propeller-shaped PI3Ki 14 , but also of leniolisib 28 .Similarly, the preceding non-conserved residue, in p110δ, regulated isoform selectivity, as exemplified by the reciprocal mutations I771Y and Y778I in p110α and p110β, respectively 47 .Since the PI3K class I isoforms p110δ and p110β at this position bear aromatic residues that permit ligand binding, as opposed to aliphatic residues in p110α and p110γ, one might speculate that the analog of the I777M mutation in p110ß (I883M) would also cause resistance to propeller-shaped p110ß-selective PI3Ki.In ZSTK474-bound p110δ, MD simulations showed a cryptic pocket between I777 and K779, the formation of which was prevented by the I777M substitution.In contrast, copanlisib bound equally well to p110δ-wt and p110δ-I777M, whereas the analogous I800M mutation in p110α mediated strong resistance.This striking difference between two isoforms in the effect of analogous mutations at a conserved affinity pocket residue is reminiscent of the role of the non-conserved residue mentioned above in isoform-specific drug affinity 47 .For three selected PI3Ki, cell-based assessment and MD simulations arrived at the same conclusions as to mutationmediated PI3Ki resistance, implying the importance of projections from the main plain of PI3Ki molecules for their interactions with N-lobe regions of p110δ.Vice versa, the I777M mutation provides a straightforward model for virtual ligand design that can be easily tested experimentally using the described cell-based systems. Taken together, mutations in p110δ enabled convenient cellbased assessment of isoform-selective PI3Ki efficacy as well as chemical genetic evaluation of p110δ-dependent cell functions and insights into the molecular interactions of PI3Ki with the binding cleft of p110δ. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Cell lines and cell culture.Ramos, BaF3, and NIH3T3 cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).TMD8 and HBL-1 cells were a gift from Dr. Georg Lenz (University of Münster).Cells were maintained in DMEM or RPMI-1640 medium supplemented with 10% fetal bovine serum and penicillin-streptomycin.The parental BaF3 cells were supplemented with 10 ng/ml IL-3.All cells were cultured at 37 °C at 5 % CO 2 . Expression constructs.PCR products containing full reading frames of human p110δ or p110α were generated from plasmid templates (Addgene #34893, 12522, 12524) for cloning into the XhoI/SacII or XhoI/NotI sites of the retroviral vector pMXs-IRES-Neo (Cell Biolabs).Mutations were introduced into the p110 reading frames on these plasmids using the QuikChange site-directed mutagenesis kit (Agilent) and the mutagenesis primers detailed in Supplementary Table 6.All PCR-generated sequences were verified by Sanger sequencing. Genetically modified cell lines.Retrovirus for transduction of BaF3 or Ramos cells was generated by liposomal transfection of pMXs-IRES-Neo expression constructs into Phoenix-Ampho cells that express retrovirus replication and amphotropic envelope proteins.For transfection, 20 µg of plasmid DNA and 60 µl of lipofectamine 3000 (Thermo Fisher) in 1.5 ml of OptiMEM (Gibco) were added to ~70 % confluent Phoenix-Ampho cells in a 10 cm cell culture dish.Following the addition of OptiMEM for 6 h and incubation in DMEM, virus-containing supernatants were collected after 72 h and used directly for infection or stored at -80 °C.Retroviral supernatants with 2 µg/ml polybrene were added to 2 × 10 5 BaF3 or Ramos cells on six-well plates for centrifugation at 800×g for 2 h.After cultivation in RPMI-1640 for 2 days, BaF3 or Ramos cells were selected with 1.5 µg/ml geneticin for 6-10 days.To verify the integration of p110δ cDNA in the genome of transduced BaF3 or Ramos cells, templates for sequence verification were amplified from isolated genomic DNA using primers with specificity for the transduced cDNA (Supplementary Table 6).In addition, we demonstrated overexpression of p110δ and expression of HA tag in Western blots. Cell viability assay.Where applicable, transduced BaF3 cells selected for vectorencoded geneticin resistance were additionally deprived of IL-3 for several days prior to viability assessment, as commonly performed in the analysis of oncogenic kinases in this system 48 .Subsequently, they were seeded on 96 wells at a density of 10 5 cells per well and grown in medium without IL-3 for 96 h.As a surrogate of viable cell numbers, ATP levels were determined using the CellTiter-Glo (CTG) assay (Promega) with luminescence measurements on a FLUOstar Optima plate reader (BMG Labtech) or metabolic activity was measured using the XTT assay (Roche). Anchorage-independent growth of fibroblasts.Colony formation in soft agar by NIH3T3 cells stably expressing p110 isoform variants was examined.Isogenic NIH3T3 cells were trypsinized and suspended at a density of 2.5 × 10 4 cells per sixwell in a medium containing 10% FBS and 0.5% agarose and plated onto a bottom layer containing 0.6% agarose.After 4 weeks of incubation, three images per well were taken at 25-fold magnification and analyzed using Image J software to determine number of colonies with areas of at least 1200 µm 2 . Drug library.PI3Ki and control substances were purchased as a custom library from Sellekchem (Munich, Germany).Leniolisib was kindly provided by Dr. Christoph Burkhart, Novartis Pharma AG, Switzerland.The compounds were solved in DMSO and stored frozen as 10 mM stocks.The concentration of DMSO as a solvent for inhibitors was kept below 0.1% in cell cultures. Drug sensitivity assessment.For drug library assessment, cells were treated with drugs in seven-point, fourfold serial dilutions ranging from 10 µM down to 2 nM that were prepared with a Felix pipetting station (Analytik Jena).With the same device, compound dilutions were transferred to white 384-well cell culture plates in a volume of 5 µl per well for four replicate measurements.Treatment with 10% DMSO was used for no-growth controls.Isogenic BaF3 cells selected for vectorencoded geneticin resistance were incubated in a medium without IL-3 for several days prior to addition to the 384-well plates with pre-dispensed inhibitors using a MultiFlo FX liquid dispenser (BioTek).Cells were seeded at a density of 10 4 cells per well (50 µl) and incubated for 72 h.Subsequently, cell viability was determined using the CTG assay.The obtained data were normalized to untreated controls and analyzed with the R package DR4PL using a four-parameter logistic model for the calculation of IC 50 values.For the final evaluation, the median of four or three library assessments was used for experiments with cells expressing variants of p110δ or p110α, respectively.With the DLBCL cell lines TMD8 and HBL-1, the drug library was assessed twice for the calculation of mean IC 50 values. Signaling analyses.Ramos cells were deprived of serum for 2 h and incubated with inhibitors for 20 min prior to the addition of 10 µg/ml of goat anti-human IgM F(ab') 2 for another 10 min.The resulting Ramos cells with activated BCR were used for signaling analyses by immune-blotting or phospho-specific flow cytometry or cytokine content of culture supernatants.The same treatment scheme was used for BaF3 cells, however using 2 h incubation with 10 ng/µl IL-3 after serum deprivation. Detection of signaling molecules in Western blots was performed using the Odyssey imager (Licor, Bad Homburg, Germany).Cell lysates containing 30 µg of total protein were immunoblotted and probed by specific primary and fluorescence-labeled secondary antibodies (Supplementary Table 7 and Supplementary Fig. 12). Cytokine quantification.Levels of the chemokines CCL3, CCL4, and TNF-α were determined in culture supernatants after stimulation of 1 × 10 6 Ramos cells/ml with anti-IgM (2 µg/mL) for 24 h by means of ELISA kits (Affymetrix, eBioscience) by means of absorbance readings at 450 nm on a FluoSTAR Optima Plate Reader.Standard curves covered concentration ranges from 16 to 2000 pg CCL3 or CCL4/ ml or 15.6 to 1000 pg TNFα/ml.Chemotaxis assay.Transwell-migration towards 100 ng/ml of the chemokine CXCL12 was determined essentially as previously described in ref. 42 .Briefly, 2 × 10 5 isogenic BaF3 cells suspended in 100 µl of medium were added to transwell culture inserts with a diameter of 6.5 mm and a pore size of 5 µm (Corning, Amsterdam, The Netherlands), that were transferred to the bottom wells containing medium with or without CXCL12 (Peprotech, Rocky Hill, NJ, USA).After 4 h of incubation, cell densities in the lower chambers were counted using a MACSquant flow cytometer (Miltenyi, Bergisch Gladbach, Germany). Molecular modeling.Human PI3Kδ crystal structure was used as a template (PDB entry 6PYR) to build a homology model formed by the helical and kinase domain using the SWISS-MODEL web-based server 49 .The model was energy-minimized by a steepest-descent algorithm in the GROMACS program 2020.4 50 .The root mean square deviation (RMSD) between the model and template is less than 0.3 Å and the quality of the model was validated with PROCHECK 51 .A single-point mutation (methionine) at residue I777 was introduced using the SCWRL4 program with default parameters 52 .As an indication of the high quality of both theoretical models, overall quality factors of 94.43 and 96.74 were obtained for p110δ-wt and p110δ-I777M models, respectively.These two structures were considered as the input structures to perform MD simulations in the next step.These two apo models (wt and mutant) were used to superimpose the coordinates of three small molecule inhibitors from the following PDB entries: idelalisib (4EX0), copanlisib (5G2N), and ZSTK474 (2WXL).A subsequent energy minimization was again performed for each system. Quantum mechanical calculations.Geometry optimization, frequency calculations, and population analyses of the three small molecule inhibitors were performed with the Gaussian 16 package of programs 53 using the B3LYP functional and the 6-31 G(d) basis set.Geometry optimization and frequency calculations were carried out in solution, using the SMD continuum model and water as solvent.SMD is considered a universal solvation model, due to its applicability to any charged or uncharged solute in any solvent or liquid medium 54 .Vibrational analysis indicates that geometries correspond to minima.Computed electrostatic potential (ESP) derived atomic charges were used later for MD simulations. Classical MD simulations.A summary of the simulations is presented in Supplementary Table 5.A total of 8 μs simulations were performed using the all-atom additive CHARMM36 protein forcefield 55 and TIP3 water model 56 with GROMACS 2020.04.Initial complexes were solvated in a rhombic dodecahedron box, with a minimum distance of 10 Å between the structure and the box boundaries.Sodium and chloride ions were added to neutralize the system to an ionic strength of 0.15 mol/L.The systems were equilibrated for 5 ns in the NVT ensemble with restrained heavy atoms, and for 5 ns in the NPT ensemble without restraints.The temperature was stabilized at 310 K using a V-rescale thermostat 57 and pressure at 1 atm by Parrinello-Rahman barostat 58 , respectively.Production MD simulations were run on a GPU (GeForce RTX 3090, Cuda 11.4), with a time step of 2 fs.Electrostatic interactions were calculated using the Particle Mesh Ewald method 59 and LINCS algorithm 60 was used for bond constraints.Input and output coordinate files of production simulations are presented in pdb format (Supplementary Data 1). Trajectory analyses.Convergence analysis of equilibrated and production simulations were performed in GROMACS (Supplementary Fig. 13a-d).Representative structures were extracted from trajectories using gmx cluster tool in GROMACS with a single linkage algorithm applying different cut-offs.The structures chosen were the center of the clusters (the structure with the smallest average root mean square deviation from all other structures of the cluster).All representative structures are presented in Supplementary Data 1.Distances and angles were calculated using GROMACS tools gmx distance and gmx angle and processed and analyzed in R 61 .Pymol was used for visualization and figure preparation 62 and PyVol 63 was used to calculate binding pocket volumes.Wilcoxon signed-rank test with a statistical significance of 0.05 was used to account for significant differences between wt and I777M systems in molecular dynamics simulations.Although we only performed a single MD simulation per condition, our simulations were able to cover a considerable timescale of microseconds.Additional replicas would be needed to ensure reproducibility. Reporting summary.Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.calculations; F.H., G.K., R.R.-R., and M.H. analyzed and interpreted data; G.K., F.H., and R.R.-R wrote the paper.All authors read, revised, and approved the paper. ",
"section_name": "Methods",
"section_num": null
}
] |
[
{
"section_content": "This study was supported by grants from the Deutsche José Carreras Leukämie-Stiftung (DJCLS R19/2016 and DJCLS 15R/2019) and the Köln Fortune program of the University of Cologne (379/2021) to G.K. and from the Exzellenz initiieren (EI)-Stiftung Kölner Krebsforschung to R.R.-R.We acknowledge support for the Article Processing Charge from the DFG (German Research Foundation, 491454339). ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Open Access funding enabled and organized by Projekt DEAL. ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "Source data for Figures can be found in Supplementary Data 2. All data supporting the findings of this study are available within the paper and its Supplementary Information. MD simulation input and output files can be found in Supplementary Data 1.The code used in the current study is mentioned in the Methods. ",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "Source data for Figures can be found in Supplementary Data 2. All data supporting the findings of this study are available within the paper and its Supplementary Information. ",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "MD simulation input and output files can be found in Supplementary Data 1.The code used in the current study is mentioned in the Methods. ",
"section_name": "Code availability",
"section_num": null
},
{
"section_content": "This study was partially supported with research funding from Gilead Sciences to M.H.The remaining authors declare no competing interests. The online version contains supplementary material available at https://doi.org/10.1038/s42003-023-04921-z. Correspondence and requests for materials should be addressed to Rocio Rebollido-Rios or Günter Krause. Peer review information Communications Biology thanks the anonymous reviewers for their contribution to the peer review of this work.Primary Handling Editors: Zhijuan Qiu and Gene Chong.A peer review file is available. Reprints and permission information is available at http://www.nature.com/reprintsPublisher'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": "This study was partially supported with research funding from Gilead Sciences to M.H.The remaining authors declare no competing 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/s42003-023-04921-z. Correspondence and requests for materials should be addressed to Rocio Rebollido-Rios or Günter Krause. Peer review information Communications Biology thanks the anonymous reviewers for their contribution to the peer review of this work.Primary Handling Editors: Zhijuan Qiu and Gene Chong.A peer review file is available. Reprints and permission information is available at http://www.nature.com/reprintsPublisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ",
"section_name": "Supplementary information",
"section_num": null
}
] |
10.3389/fped.2023.1224966
|
Genomic signatures and prognosis of advanced stage Chinese pediatric T cell lymphoblastic lymphoma by whole exome sequencing
|
<jats:sec><jats:title>Objective</jats:title><jats:p>To investigate the genomic signatures and prognosis of advanced-stage T cell lymphoblastic lymphoma (T-LBL) and to examine the relationship between T-LBL and T cell acute lymphoblastic leukemia (T-ALL).</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>35 Chinese T-LBL children with stage III or IV disease were recruited for this study. They were treated with combination chemotherapy and whole exome sequencing. The relationship of the clinical features, prognosis and specific gene mutations was researched. Gene chips of T-LBL and T-ALL were downloaded from a database, and differential gene expression was analyzed.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Germline causal gene mutations (CARS or MAP2K2) were detected in 2 patients; 3.06 ± 2.21 somatic causal gene mutations were identified in the 35 patients, and somatic mutations were observed in the NOTCH1, FBXW7, PHF6 and JAK3 genes. NOTCH1 mutations were significantly associated with FBXW7 mutations, and the age at diagnosis of patients with NOTCH1-FBXW7 mutations was less than that of patients without such mutations (<jats:italic>P</jats:italic> &lt; 0.05). 32 patients achieved complete remission (CR), and 14 and 18 patients were classified into the intermediate risk (IR) group and high risk (HR) group. During a median follow-up of 44 months, 3 patients relapsed. Three-year prospective event free survival (pEFS) was 82.286%, and no significant differences of pEFS were found for different sexes, ages, or statuses of NOTCH1-FBXW7 mutations, (<jats:italic>P</jats:italic> &gt; 0.05); however, the mean survival time of the IR group was longer than that of the HR group (<jats:italic>P</jats:italic> &lt; 0.05). Differential expression of genes in the T-LBL and/or T-ALL datasets was analyzed using the R package limma, and 1/3 of the differentially expressed genes were found in both the T-ALL and T-LBL datasets. High expression of PI3K-Akt signal pathway genes and the USP34 gene was found in the T-LBL dataset.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>Although T-ALL and T-LBL both originate from precursor T-cells and are considered different manifestations of the same disease and the outcome of T-LBL is favorable when using T-ALL-based chemotherapy, there are differences in the gene distribution between T-LBL and T-ALL. It seems that the PI3K-Akt signaling pathway and the USP34 gene play important roles in T-LBL, but medicines targeting the USP34 gene or the PI3K-Akt pathway may be invalid.</jats:p></jats:sec>
|
[
{
"section_content": "Lymphoblastic lymphoma (LBL) is an aggressive non-Hodgkin's lymphoma (NHL) that accounts for 20%-30% of the pediatric NHL population (1).T cell LBL (T-LBL) involves the precursor T-cell immunophenotype and represents 70%-80% of LBL cases in children (1,2).The prognosis of T-LBL in children has remained poor historically.Acute lymphoblastic leukemia (ALL)-based chemotherapy and allogenic hematopoietic stem cell transplantation have demonstrated effective results, and eventfree survival (EFS) exceeds 80%-90% even for advanced stage (stage III and IV) patients (2,3), but the survival of relapsed and/or refractory cases remains poor (<10%-30%). Gene mutations, such as FBXW7, NOTCH1, and PTEN mutations, play important roles in T-LBL development and are potential prognostic biomarkers for T-LBL (2,4).Pathogenetic and molecular biological studies of T-LBL are limited due to the limitations of detection methods and sufficient available materials. Next-generation sequencing (NGS) technologies have become the method of choice for gene mutation analysis in cancer (4,5), but NGS data from Chinese pediatric T-LBL patients have not yet been reported.In the current study, whole exome sequencing (WES) by NGS was used to analyze T-LBL patients with the aim of gaining detailed genome-wide insight into the signature and mechanisms of tumorigenesis in pediatric T-LBL.The relationship of prognosis and specific gene mutations in children with T-LBL was also researched.Gene chips were downloaded from a database, and differential gene expression was analyzed. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Methods",
"section_num": "2."
},
{
"section_content": "Patients with newly diagnosed T-LBL admitted to Children's Hospital of Chongqing Medical University (CHCMU) and Children's Hospital of Xianyang (CHX) between January 2013 and September 2020 were enrolled in the study.Diagnosis of T-LBL was in accordance with the World Health Organization (WHO) criteria of 2008 or 2016 (6,7), and patients were staged with the revised international pediatric NHL staging system (IPNHLSS) (8).Patients who were ≥18 years at diagnosis, diagnosed with mixed-phenotype LBL or secondary lymphoma, or had human immunodeficiency virus infection were excluded; patients who were classified with a local stage (stage I or II) and who had received chemotherapy before hospitalization were also excluded from the study. Pathologic diagnosis of T-LBL patients was confirmed by lymph node biopsy.Immunohistochemical staining was detected as described in the literature (7); fluorescence in situ hybridization (FISH) of MYC, MLL and SIL-TAL was also performed (9,10).Blast cells from bone marrow (BM) samples <25% was defined as the cutoff value between T-LBL (stage IV) and T-ALL (1,6). Patients were treated with the modified LBL-1995 Berlin-Frankfurt-Münster protocol (BFM-LBL-95) (4,11).An intrathecal injection was administered as the protocol required, and cranial radiotherapy was carried out for patients with central nervous system (CNS) involvement.Treatment response was evaluated at three time points (TP): TP1 or TP2 (days 15 or 33 of remission induction) and TP3 (prior to consolidation).Minimal disseminated disease (MDD) levels were detected in BM samples using computed tomography (CT) or positron emission tomography computed tomography (PET-CT) screening as described in previous literature reports, and disease status was evaluated as partial remission (PR), complete remission (CR), progressive disease (PD) or refractory disease (RD) (12)(13)(14).All patients with advanced stage were regarded as the intermediate risk (IR) group at diagnosis; patients who presented with PD at TP1, PR at TP1 and CR at TP2 were considered as the high risk (HR) group; patients who did not achieve CR at TP3 were considered as having RD. The details of the risk group classification, course of treatment and drug dosage for the modified BFM-LBL-95 protocol are listed in the Supplementary Material.Clinical data, laboratory findings and prognosis data of enrolled patients were collected and analyzed retrospectively. ",
"section_name": "Patients and treatment",
"section_num": "2.1."
},
{
"section_content": "Tumor DNA samples of T-LBL patients were obtained at diagnosis from formalin-fixed specimens; germline samples were collected from the oral mucosa of patients and their parents' peripheral blood (PB).Genomic DNA was extracted using a QIAmp DNA Minikit (QIAGEN, China).Genomic DNA was enriched, and sequencing was carried out (Agilent SureSelect Human All Exon V6).PCR products of the whole exome were sequenced (Illumina HiSeq PE 150 bp). The original WES sequencing data were read using Illumina Pipeline software (version 1.3.4),and data were obtained from databases (dbSNP, 1,000 Genomes Project, ClinVar, ESP6500, ExAc, Ensembl, HGMD, UCSC, etc.).Mutated genotypes were determined using GATK, LRT, Mutation Taster and SamTools software. The identified variants were divided into the following four categories according to previous literature reports (15) and software analysis: (1) pathogenic genotypes that were confirmed by literature reports; (2) likely pathogenic genotypes that were reported in literature reports and/or affected proteins by function prediction; (3) indefinite variants and (4) single nucleotide polymorphisms (SNPs) or single nucleotide variants (SNPs).Pathogenic genotypes and likely pathogenic genotypes were recorded as causal gene mutations, and causal gene mutations of tumor samples were confirmed by Sanger sequencing.Germline samples were cross-checked and detected by Sanger sequencing, and causal somatic or germline gene mutations were identified. ",
"section_name": "DNA isolation and sequencing",
"section_num": "2.2."
},
{
"section_content": "The gene chip dataset GSE29986 was downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/).These datasets included 20 T-LBL samples, 10 T-ALL samples and 6 ALL samples.Differential expression analysis was performed using the R package limma.First, we performed differential analysis of the T-LBL and T-ALL datasets, and we obtained upregulated and downregulated genes in the datasets (adj.P. Val <0.05 and | logFC|≥1).Next, we performed differential analysis of the T-LBL and ALL datasets, and we identified upregulated and downregulated genes in these two datasets using the same filtering threshold.Volcano plot showing differentially expressed genes (DEGs) was generated using R language ggplot2.We determined the overlapping genes from the previous two rounds of differential analysis.The overlapping upregulated and downregulated genes related to T-LBL were selected for downstream analysis. ",
"section_name": "Identification of DEGs between the T-LBL and T-ALL/ALL datasets",
"section_num": "2.3."
},
{
"section_content": "We used R package weighted gene coexpression network analysis (WGCNA) to construct coexpression modules.Thirty-six samples were used to calculate Pearson's correlation coefficients.A power of 6 was selected.An unsigned hybrid coexpression network was then generated using the standard settings.We selected 5,000 genes to construct a topological heatmap.We performed Pearson correlation analysis between the module eigengenes and the trait data to identify module-trait relationships.Finally, we selected turquoise module (related to T-LBL) genes to construct a gene regulatory network and performed Gene Ontology (GO) enrichment analysis. ",
"section_name": "WGCNA of the 36 samples",
"section_num": "2.4."
},
{
"section_content": "The R package clusterProfiler was used to analyze the GO enrichment of the upregulated gene and downregulated genes related to T-LBL.Biological process (BP) analysis, cellular component (CC) analysis, molecular function (MF) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the selected genes were carried out.After performing WGCNA, the hub genes related to T-LBL were selected as input genes for BiNGO.We used Cytoscape software to visualize the results. ",
"section_name": "GO and KEGG pathway enrichment analyses",
"section_num": "2.5."
},
{
"section_content": "To verify the hub genes (USP34, C3 and MGP) identified in WGCNA, we explored the expression level of the hub genes in TCGA (tumor datasets).We checked the expression level of a hub gene (USP34) in different cancers in the CCLE database (https://portals.broadinstitute.org/ccle/home). ",
"section_name": "Validation of hub gene expression with TCGA and CCLE databases",
"section_num": "2.6."
},
{
"section_content": "Events were defined as each of the following situations (2, 4): RD at TP3, relapsed disease, death or diagnosis of a secondary malignancy, or loss to follow-up.With follow-up to December 2020, data on the clinical features, laboratory findings, WES sequencing, treatment responses, CR rate, treatment-related mortality (TRM) and prospective event-free survival (pEFS) of the patients were collected and analyzed. EFS was calculated from the date of diagnosis to the last followup, loss of follow-up or first event.SPSS 19.0 (IBM Corp., Armonk, NY) software was applied for statistical analysis.Survival curves were calculated according to the Kaplan-Meier test.Proportional differences between patient groups were analyzed by Pearson chisquared (χ 2 ) tests or Fisher's exact tests.A P value <0.05 was regarded as a significant difference. ",
"section_name": "Statistical analysis",
"section_num": "2.7."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "Sixty patients with newly diagnosed T-LBL were admitted in the study period; 2 patients were classified as stage I or stage II, and chemotherapy was refused by 4 patients because of family choice.Twelve patients received chemotherapy, but WES sequencing failed because of insufficient available tumor samples.WES sequencing was refused by 7 families.Thirty-five patients received chemotherapy, and WES sequencing was also performed. The 35 enrolled patients included 25 patients with stage III disease and 10 patients with stage IV disease; 26 males and 9 females were included; family members with gastric cancer were found for 2 (5.71%) patients.Age at diagnosis was 15-168 m (median value: 86 m; average value: 92.83 ± 45.09 m); serum level of lactate dehydrogenase (LDH) was 180-1,947.1 U /L (median value: 339.6 U / L, average value: 522.14 ± 462.40 U /L, normal value <220 U /L), ≥twofold normal level of LDH was detectable in 16 (45.71%)patients; mediastinal, BM or testicular involvement was found in 32 (91.43%), 10 (28.57%) or 1 (2.86%) patients, respectively; and CNS involvement was undetectable in the cohort (Table 1). ",
"section_name": "Clinical and laboratory characteristics",
"section_num": "3.1."
},
{
"section_content": "WES sequencing of the 35 patients was performed, and germline causal gene mutations (CARS or MAP2K2) were detected in 2 patients without a family history of cancer; 1-13 (average 3.06 ± 2.21; median 2) somatic causal gene mutations were identified in the 35 patients, and the somatic mutations were observed in the NOTCH1, FBXW7, PHF6 and JAK3 genes (Figure 1A).The relationship among these gene mutations was demonstrated by corplot (Figure 1B), and NOTCH1 mutations were significantly associated with FBXW7 mutations (6/35, 17.14%).The associations between NOTCH1-FBXW7 mutational status and clinical characteristics are listed in Table 2, which revealed that the age at diagnosis of patients with NOTCH1-FBXW7 mutations was less than that of patients without such mutations (P < 0.05), whereas significant differences were not found for sex distribution, LDH level or disease staging distribution (P > 0.05).These results were similar to those for pediatric T-ALL in previous literature reports (16,17). ",
"section_name": "Results of WES sequencing",
"section_num": "3.2."
},
{
"section_content": "Treatment effects were evaluated at different TP levels as the protocol required.Twelve, 21 or 2 patients were classified as CR, PR or PD at TP1 and TP2, respectively; at TP3, 1 PR patient died of sepsis, 32 achieved CR, and 2 patients were classified as PD, and the CR rate was 91.43% after they finished the course of remission induction.The 2 PD patients quit the study, and 14 and 18 patients were classified into the intermediate risk (IR) group or high risk (HR) group; chemotherapy was continued with the different risk group strategy (Table 3).With follow-up to December 2020, 3 patients relapsed.Among the 35 patients in the cohort, TRM was 2.86%, the relapse rate was 8.57%, and 3-year pEFS was 82.286% (95% CI 64.71-91.64%,Figures 2A,B).pEFS was compared among different sexes, ages, LDH levels and disease stages, but no significant differences were found (P > 0.05, Figures 2C-E). The 35 patients were classified into 2 groups (group A: NOTCH1-FBXW7 mutation; group B: without the NOTCH1-FBXW7 mutation), and the treatment responses and outcomes were also calculated with different mutational statuses.There was no significant difference in the CR rate between the two groups at TP1, TP2 or TP3 (P > 0.05, Table 4).One patient in each group remained PD at TP2; the remaining patients were treated as different risk groups, and a significant difference was not found between different mutational statuses or risk groups (P > 0.05, Table 4, Figures 2F-H), but the mean survival time of the IR group was longer than that of the HR group (66.14 ± 7.88 vs. 42.07 ± 8.46 m, P < 0.05).Literature reports have shown that the NOTCH1-FBXW7 mutation is related to favorable outcomes (18-20), but our study showed that the diversity of responses might be overcome by intensive chemotherapy; however, a larger sample and multiple centers are needed to verify this hypothesis. ",
"section_name": "Treatment evaluation and prognosis",
"section_num": "3.3."
},
{
"section_content": "To further understand the pathophysiolopoiesis, differences and similarities of T-LBL and T-ALL, differential expression analysis was performed, and the results were visualized.A volcano plot was generated to show the results of the differential expression analysis; a Venn plot was generated to show the overlapping upregulated and downregulated genes from the differential expression analysis (T-LBL versus T-ALL/ALL, Figures 3A,B).Although T-ALL and T-LBL are considered different manifestations of the same disease ,D).The overlapping upregulated genes or downregulated genes related to T-LBL were assessed by GO and KEGG pathway enrichment analyses (BP, CC and MF), and the interactions between the gene sets and GO terms were analyzed and visualized; it appeared that the PI3K-Akt signal pathway, focal adhesion and ECM-receptor interaction play roles in the pathophysiology of T-LBL (Figure 4).However, further studies are required to confirm this result.Sample hierarchical clustering plots and trait heatmaps from the GSE29986 dataset were generated, and the topological overlap matrix (TOM) of coexpressed genes in different modules of the top 5,000 genes is shown as a heatmap (Figure 5).Module and trait relationships were determined, and the turquoise module was the most related module to TLBL; the coexpression network of significant genes in the turquoise module (related to T-LBL) was generated (Figure 5).The mRNA expression levels of three key genes (USP34, C3 and MGP) based on TCGA tumor data were also determined, and the USP34 gene was highly expressed in the cancer cell lines included in the T-LBL and T-ALL datasets according to CCLE analysis (Figure 6). Ubiquitination is a common posttranslational modification of proteins and is involved in many physiological activities, such as cell division and differentiation, growth and development, transcriptional regulation, injury stress, and immune response (23).Deubiquitination enzymes have diverse structures and functions; they can remove ubiquitination modifications, affect protein function and regulate physiological activities.The ubiquitin-specific peptidase (USP) superfamily is one of the most widely known deubiquitinase families with the most diverse structures, and the USP34 gene encodes a member of the USP family that can remove ubiquitin molecules from large protein molecules and eliminate the biological functions of ubiquitinated proteins (24,25).This USP plays a key regulatory role in the ",
"section_name": "Identification of DEGs between the T-LBL and T-ALL/ALL datasets",
"section_num": "3.4."
},
{
"section_content": "T-LBL is an aggressive form of non-Hodgkin's lymphoma derived from precursor T cells and makes up approximately 80% of the LBL population.T-ALL is regarded as the leukemic phase of T-LBL (1,2).Several studies have demonstrated that leukemia-based therapy (e.g., the BFM protocol series) and allo-HSCT are effective and lead to a good prognosis for T-LBL patients, and EFS exceeds 80%-90% for advanced stage patients in developed or developing countries (2,3,20,28).Traditional prognostic factors have been challenging because age, sex, and multiple organ involvement have not been found to be prognostically significant by the literature (2, 3) or our study.Germline mutations (CARS or MAP2K2 mutation) were detected in 2 patients without cancer history and the relationship between tumor development and hereditary should be considered, but data were limited, further data were needed to investigate it. Multiple gene mutations play different roles in the development, progression and carcinogenesis of T-LBL and T-ALL.Our data and the literature suggest that the common somatic mutations are NOTCH1, FBXW7 and PHF6 mutations and that NOTCH1 mutations are significantly associated with FBXW7 mutations (5,16,20,29).The associations between NOTCH1-FBXW7 mutational status and clinical characteristics are listed in Table 2, which revealed that the age at diagnosis in patients with NOTCH1-FBXW7 mutations was less than that in patients without such mutations (P < 0.05), whereas significant differences were not found for sex distribution, LDH level or analyses, and it appeared that the PI3K-Akt signaling pathway and USP34 gene played important roles in T-LBL.Tumor development, proliferation and metastasis are regulated by abnormal cellular signaling pathways, and genes in the phosphoinositide 3-kinase (PI3K)/Akt pathway are the most frequently altered in human cancers.Aberrant activation of this pathway is associated with cellular transformation, tumorigenesis, cancer progression, and drug resistance (31,32).The ubiquitin-specific protease 34 (USP34) gene and its protein are closely related to development and progression of human cancers.Research has shown that USP34 overexpression can be detected in diffuse large B cell lymphoma instead of follicular lymphoma, but a significant association has not been identified between the USP34 protein level and patient survival (33,34).Pancreatic cancer cell proliferation and migration are promoted by USP34 overexpression via upregulation of the Akt protein, and pancreatic cancer cell apoptosis induced by an Akt inhibitor is reversed by USP34 overexpression (35).Thus, we assume that USP34 overexpression and the PI3K-Akt signaling pathway are associated with T-LBL development and progression, but anticancer therapies targeting the USP34 gene or the PI3K-Akt signaling pathway may be invalid for refractory relapsed T-LBL patients.Further laboratory studies should be performed. ",
"section_name": "Discussion",
"section_num": "4."
}
] |
[
{
"section_content": "We thank the patients and their families who participated in this study.The authors declare that there are no competing interests associated with the manuscript. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "The study was supported by the National Natural Science Foundation of China (Project No. 81900162) and the Chongqing Science and Technology Commission of the PR China (Project No. cstc2018jsyj-jsyjX0015); the funding sources had no roles in the design and conduct of the study collection; management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and the decision to submit the manuscript for publication.The study was registered at the Chinese Clinical Trail Registry and the clinical study registration (ChiCTR1900025690 and ChiCTR-IPR-14005706). ",
"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 in the article/Supplementary Material. ",
"section_name": "Data availability statement",
"section_num": null
},
{
"section_content": "The studies involving humans were approved by Ethics Administration Office of Children Hospital of Chongqing Medical University.The studies were conducted in accordance with the local legislation and institutional requirements.Written informed consent for participation in this study was provided by the participants' legal guardians/next of kin. JX conceived and designed the study.QL, XY and NY prepared the figures and tables.QL, XY, JW, XL, PZ and YG analyzed and interpreted the data.LS and JX drafted the manuscript.JX revised 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. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fped.2023.1224966/full#supplementary-material ",
"section_name": "Ethics statement",
"section_num": null
},
{
"section_content": "The studies involving humans were approved by Ethics Administration Office of Children Hospital of Chongqing Medical University.The studies were conducted in accordance with the local legislation and institutional requirements.Written informed consent for participation in this study was provided by the participants' legal guardians/next of kin. ",
"section_name": "Ethics statement",
"section_num": null
},
{
"section_content": "JX conceived and designed the study.QL, XY and NY prepared the figures and tables.QL, XY, JW, XL, PZ and YG analyzed and interpreted the data.LS and JX drafted the manuscript.JX revised 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
},
{
"section_content": "The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fped.2023.1224966/full#supplementary-material ",
"section_name": "Supplementary material",
"section_num": null
}
] |
10.3389/fonc.2021.637186
|
TH2/TH1 Shift Under Ibrutinib Treatment in Chronic Lymphocytic Leukemia
|
<jats:p>Ibrutinib may revert the T-helper (Th)2 polarization observed in chronic lymphocytic leukemia (CLL) by targeting the IL-2-inducible kinase, that shows a significant homology with the Bruton tyrosine kinase. In the front-line GIMEMA LLC1114 trial (ibrutinib+rituximab for 6 months, followed by ibrutinib maintenance), we investigated the modulation of T-cell cytokine production in 208 peripheral blood paired samples from 71 CLL patients: 71 samples prior to treatment (Day 0, D0) and at day +14 (D14; n=50), at month +8 (M8; 30), +12 (M12; 25), +18 (M18; 22) and +24 (M24; 10) of treatment. We documented a progressive decrease of CD3+CD4+IL-4+ T cells (Th2), that was significant at M8 and at M12 (p=0.019, p=0.002), a relative increase in the CD3+CD4+IFNγ+ T cells (Th1) and a decrease of CD3+CD4+IL-17+ (Th17) cells that was maintained up to M18 (M8 <jats:italic>vs</jats:italic> D0 p=0.003, M12 <jats:italic>vs</jats:italic> D0 p=0.003, M18 <jats:italic>vs</jats:italic> D0 p=0.004) of ibrutinib treatment. The Th2/Th1 ratio significantly decreased already after 14 days of treatment and was maintained thereafter (D14 <jats:italic>vs</jats:italic> D0 p=0.037, M8 <jats:italic>vs</jats:italic> D0 p=0.001, M12 <jats:italic>vs</jats:italic> D0 p=0.005, M18 <jats:italic>vs</jats:italic> D0 p=0.002). The Th2/Th1 modulation over time was significant only among patients with unmutated IGHV. The Th2/Th1 ratio below a cut-off of 0.088 at M8 was associated with the achievement of a complete response (CR) (p=0.016). Ibrutinib may shape the CLL T-cell profile, limiting Th2 activation and inducing a shift in the Th2/Th1 ratio. The association between the Th2/Th1 ratio decrease and the CR achievement suggests the <jats:italic>in vivo</jats:italic> generation of a potential host anti-tumor immune activation induced by ibrutinib.</jats:p>
|
[
{
"section_content": "In chronic lymphocytic leukemia (CLL), malignant B cells and the surrounding tissue microenvironment closely interact.The complex cross talk between normal and CLL cells plays a critical role in the survival, growth and drug resistance of leukemic cells (1)(2)(3).In addition, the leukemic clone develops strategies of evasion or suppression of the immune system, in particular toward the anti-tumor effects of T lymphocytes (4)(5)(6)(7). The T-cell compartment in CLL patients is dysfunctional, showing in particular: i) an increased expression of inhibitory receptors and a defective immune synapse formation upon contact with CLL cells (8); ii) skewing of T-cell subsets from naïve to memory T cells and an increase in the number of circulating T cells both in the CD4 + and the CD8 + compartments (9); iii) a higher expression of exhaustion markers (10); iv) within the CD4+ cells, a T-helper (Th)2/Th1 ratio imbalance with an aberrant recruitment of a Th2-dominant response (11,12). Indeed, CLL cells produce IL-6 and stimulate IL-4 production by T cells, skewing the immune system toward a Th2-phenotype, that releases IL-4, IL-5, IL-10 and IL-13 (13)(14)(15)(16).Moreover, IL-10, a cytokine produced by Th2 cells, is a powerful inhibitor of the Th1 cytokine synthesis -including interferon g (IFNg), tumor necrosis factor a (TNFa), IL-2 and lymphotoxin (LTa) (14) and stimulates B-cell proliferation and differentiation, thus promoting the skewing toward a Th2 response. Finally, CLL cells also contribute to the imbalance between Th17 and T regulatory cells (T regs) by producing IL-10 or TGF-b, which promote the development of Tregs and suppress Th1, Th17 and cytotoxic T-cell responses.An increase in the number of Th17 cells (able to produce IL-17A and IL-17F) (17,18) has been associated with an improved survival of CLL patients, while a decreased frequency of Th17 cells has been generally found to be associated with a Tregs expansion and disease progression (19). The constitutive activation of the B-cell receptor (BCR) is central to CLL pathogenesis and prognosis.Among the new generation of biologic drugs, ibrutinib, an irreversible inhibitor of the Bruton tyrosine kinase (BTK), involved in the downstream pathway of the BCR signaling, has demonstrated outstanding clinical activity and tolerability in CLL (20).The IL-2-inducible kinase (ITK) is a T-cell dominant member of the TEC kinase family that drives proximal T-cell receptor (TCR) signaling, resulting in cellular activation, cytokine release and rapid proliferation (21)(22)(23).ITK plays a key role in the activation of Th1, Th2 and Th17 cells.In Th1 cells, ITK signaling is supportive but dispensable due to the redundant resting lymphocyte kinase (RLK) signaling, whereas in Th2 cells ITK signaling is essential for activation (23).The significant homology between BTK and ITK supports the role of ibrutinib as an immunomodulatory inhibitor of both BTK and ITK (24).Indeed, and at variance from chemoimmunotherapy, ibrutinib mitigates the immune dysregulation induced by CLL, by modifying the absolute number of T-cells (25)(26)(27)(28), the T-cell receptor repertoire (26,29), the T-cell maturation (27,28), the Th1 and Th2 polarization (24)(25)(26)(27), the Th17 and Treg cell balance (25,27,28), the T cell inhibitory receptors and function (25,(27)(28)(29).These effects, all modulated toward an anti-tumor T-cell response, have been nicely reviewed (30)(31)(32).Regarding the Th1 and Th2 polarization, it has been shown that ibrutinib may contribute to revert the Th2-dominant response observed in CLL, thus influencing T-cell mediated cancer immune surveillance, in vitro and in a mouse model (24) and in patients treated with ibrutinib (25)(26)(27).However, the variability of patients' selection and of the experimental assays makes the results not always consistent.Differences between the reported in vitro and in vivo effects of ibrutinib therapy on Th polarization highlights the importance of correlative analysis in clinical trials. In order to gain insights into the role of ibrutinib on the Th2/ Th1 balance in vivo, we investigated the modulation of T-cell cytokine production in CLL patients enrolled in the ibrutinib + rituximab front-line GIMEMA LLC1114 trial. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "Cytokine production was evaluated in patients enrolled in the front-line GIMEMA LLC1114 trial (NCT02232386) for treatment-naive (TN) and unfit CLL patients requiring treatment according to the 2008 revised iwCLL criteria (33).The protocol was based on the administration of ibrutinib (420 mg/die) + rituximab (375 mg/m 2 ), weekly in the first month and monthly from the 2 nd to the 6 th month) for 6 months, followed by ibrutinib maintenance, given up to 6 years or until disease progression, toxicity or undetectable minimal residual disease (MRD) for 6 months. To follow intra-individual variations, Th-cell cytokine production was evaluated longitudinally in 71 patients, 25 females and 46 males; median age was 72.6 years (range 37.2-86.6).A total of 208 peripheral blood (PB) paired samples was included: 71 were collected prior to ibrutinib treatment (Day 0, D0), 50 after 14 days (D14), 30 after 8 months (M8), 25 after 12 months (M12), 22 after 18 months (M18) and 10 after 24 months (M24). All patients gave a written informed consent for the research purposes according to the Declaration of Helsinki.The diagnosis of CLL was based on the current guidelines (33). ",
"section_name": "Patients' Samples",
"section_num": null
},
{
"section_content": "PB mononuclear cells (PBMC) were obtained by centrifugation on a Ficoll-Histopaque (Axis-Shield, Oslo, Norway) gradient.For intracytoplasmic detection of IFNg, IL-4 and IL-17 producing T cells, freshly isolated PBMC were initially stimulated for 5 hours in a classic culture medium with phorbol myristate acetate PMA (50 ng/ml) (Sigma-Aldrich, St. Louis, MO) plus ionomycin (1µg/ml) (Sigma-Aldrich) in the presence of Golgi-Stop (supplemented in the Human Th1/Th2/ Th17 Phenotyping Kit, Becton Dickinson (BD), San Jose, CA).Activated T cells were washed twice in PBS, divided into tubes and then fixed, permeabilized and stained using the Human Th1/ Th2/Th17 Phenotyping Kit following the manufacturer's instructions.Finally, cells were washed and analyzed by flow cytometry on a FACSCanto I flow cytometer (BD).For each analysis, 100,000 events were acquired, and analyzed using the FACSDiva software (BD), as detailed in Figure 1.In particular, lymphocytes were initially gated according to forward and side scatter features.An additional gate was established for CD4+ cells.The Th2, Th1 and Th17 cells results were reported as percentages of the CD4+ lymphocyte population.The absolute number of lymphocyte sub-populations was calculated by multiplying the immunophenotype percentages by the absolute number of lymphocytes obtained from the full blood count of the same sample. ",
"section_name": "Intracellular Cytokine Production Assay",
"section_num": null
},
{
"section_content": "Patients characteristics were summarized by means of crosstabulations for categorical variables or by means of median and interquartile range (IR) for continuous variables.Nonparametric tests have been performed for comparisons between groups (Chi-Squared and Fisher Exact test in case of categorical variables, Wilcoxon and Kruskal-Wallis test in case of continuous variables).Paired samples were compared in all the different timepoints respect to the baseline (D14 vs D0, M8 vs D0, M12 vs D0, M18 vs D0, M24 vs D0) using the Wilcoxon test.Pearson's correlation test was used to evaluate the association between continuous variables.The Th2/Th1 ratio levels over time was assessed using the Cochran-Mantel-Haenszel test.Receiver operating characteristic (ROC) curve was constructed to identify the best predictive Th2/Th1 ratio threshold (cut-off value) capable of correlating with clinical response to ibrutinib.All tests were 2-sided, accepting p<0.05 as indicating a statistically significant difference.All analyses were performed using the SAS software (release 9.4) and R statistical software (version 3.6.1). ",
"section_name": "Statistical Analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "To follow intra-individual variations, we performed a longitudinal analysis comparing paired samples from the same patient at baseline and at subsequent time points during ibrutinib treatment. We documented, as expected, a significant decrease in the percentage of CLL cells after 8 months of treatment (M8) that was maintained thereafter (Wilcoxon paired test M8 vs D0, M12 vs D0 and M18 vs D0, p<0.0001, each; M24 vs D0, p=0.005) (Figure 2A).Similar results were observed when analyzing the absolute number of CLL cells that showed an initial but not significant increase in the PB at D14 (Figure 2B). We observed a corresponding significant rise in the percentage of CD4 + T cells that was retained during in vivo ibrutinib treatment (M8 vs D0, M12 vs D0 and M18 vs D0, p<0.0001, each; M24 vs D0 p=0.012) (Figure 2A, Figure S1).The absolute numbers of CD4 + T cells showed a significant increase compared to pre-treatment samples at D14 (p=0.023),followed by a subsequent decrease starting from M8, that became significant at M12 (p=0.037)(Figure 2B, Figure S2). Furthermore, we detected a progressive increase in the percentage of CD3 + CD4 + IFNg + T cells (Th1), with a decrease of CD3 + CD4 + IL-4 + T cells (Th2) cells.In particular, Th1 cells increased from 17% at D0 (IR 12.1-26.2%)to 28.7% (IR 21.1-38.8%)at M12, reaching a significant difference from D0 only at M18 (p=0.03).Th2 cells were significantly reduced from M8 and remained so at M12 and M18 (M8 vs D0 p=0.007,M12 vs D0 We also evaluated the variations of the Th1, Th2 and Th17 Tcell subsets in terms of absolute cell counts (Figure 3B, Figure S2).In line with the data obtained analyzing the percentages of cytokine producing T cells, a significant decrease in the absolute number of activated Th2 and Th17 cell subsets was recorded.In particular, circulating Th2 cells count diminished significantly at M8 and at M12 (p=0.019;p=0.002 respectively), while the absolute number of Th17 cells decreased significantly at M8 and was maintained up to M18 of ibrutinib therapy (M8 vs D0 p=0.003,M12 vs D0 p=0.003,M18 vs D0 p=0.004).On the other hand, the absolute Th1 cell count, after a significant increase at D14 of ibrutinib treatment (p=0.025), at M8 was restored as at pre-treatment conditions (D0=192/µl; M8=176/µl) with a subsequent slight increase up to M18, although not significant (Figure 3B, Figure S2). The relative and absolute amount of CLL, CD4 + T cells, Th1, Th2 and Th17 cells recorded at D0 and at subsequent time points are shown in the supplementary material (Tables S1A, B, S2A, B). The shift toward a type 1 or type 2 cytokine producing cells was expressed more effectively by determining the ratio of Th2 to Th1 cells, allowing us to monitor through a single value the T-cell subset modulation upon ibrutinib.The median Th2/Th1 ratio at baseline was 0.19 (range 0.02-2.59); it was <1 in 90% (n=64) and ≥1 in 10% (n=7) of cases.At the subsequent time points, the Th2/Th1 ratio showed a significant decrease already after 14 days of ibrutinib treatment, that was maintained up to M18 (Wilcoxon paired test: D14 vs D0 p=0.037,M8 vs D0 p=0.001,M12 vs D0 p=0.005,M18 vs D0 p=0.002) (Figure 4, Figure S1, Table S1B).At M18, 17/22 (77%) patients showed a decreased in the Th2/Th1 ratio. To understand which of the Th1 or Th2 cells play a dominant role in the decrease of the Th2/Th1 ratio, we performed a correlation analysis of Th1 and Th2 cell modulation on the Th2/Th1 ratio at D0 and at M8 (Tables S3A,B).At both time points, the decrease of the Th2/Th1 ratio was sustained by the significant reduction of Th2 cells (p<0.0001each), corresponding to a reversion of the Th2 dominant response observed in CLL. ",
"section_name": "Longitudinal Analysis of T-Cell Cytokine Production Under Ibrutinib Treatment",
"section_num": null
},
{
"section_content": "We explored potential relationships between the modulation of Th1, Th2 and Th17 cells, and CLL clinico-biological features (Table S4).Interestingly, the Cochran-Mantel-Haenszel test showed that the decrease of the Th2/Th1 ratio over time was significant only among CLL cases with unmutated IGHV genes (p<.0001) in the cohort of patients with paired samples (30 patients with both D0 and M8, 19 with unmutated IGHV and 11 with mutated IGHV (Figure 5).These data hold true also in the total cohort of evaluated patients with paired and unpaired samples: 82 patients, 52 with unmutated IGHV and 30 with mutated IGHV (data not shown). No significant correlations between the Th2/Th1 ratio and other clinico-biologic characteristics of CLL patients were found, possibly due to the low number of patients belonging to specific In order to explore a potential relationship between the modulation of Th1 and Th2 and the response of CLL to ibrutinib, we stratified the Th2/Th1 ratio at various time points according to the clinical response at M8: complete response (CR) or no complete response (no CR).This analysis showed a significant correlation between a lower Th2/Th1 ratio at baseline (p=0.05) and at M8 (p=0.023), and the achievement of a CR (Table S5).M18 and M24 were not included in this analysis because of the small number of samples. In 30 patients evaluated both at D0 and at M8, a ROC analysis showed a correlation between the decrease of the Th2/Th1 ratio below a cut-off value of 0.088 and the achievement of a CR (p=0.016) at M8 (Figure 6).On the basis of this cut-off, 20 of the 30 patients (66.7%) were classified as high Th2/Th1 ratio group (Th2/Th1 >0.088) and 10 (33.3%) as low Th2/Th1 ratio group (Th2/Th1 <0.088).In the low Th2/Th1 ratio group, 4 of the 10 patients achieved a CR, while no patient with a Th2/Th1 ratio over the cut-off did.The decrease of Th2/Th1 ratio <0.088 at M8 also significantly correlated with the decrease of CLL cells (p=0.025)(Table S6). No correlation between Th2/Th1 modulation and the occurrence and type of infections was recorded, due to the low number of infectious events and their early occurrence in the cohort of analyzed patients. However, a significant correlation between the Th2/Th1 decrease and an IgA increase at M12 was found (data not shown), as recently suggested (34). ",
"section_name": "Modulation of T-Cell Cytokine Production, CLL Biology and Response to Ibrutinib",
"section_num": null
},
{
"section_content": "The effect of ibrutinib on the T-cell polarization and the induced revertion of the Th2-dominant response observed in CLL has been demonstrated in vitro and in mouse models by Dubovsky et al. (24).A shift toward the Th1 phenotype was detected in healthy donor and CLL T cells treated with ibrutinib in vitro.In a mouse model of leishmania infection, treatment with ibrutinib promoted a Th1-type inflammatory response with clearance of the parasite (24). In the present study, we evaluated by flow cytometry the modulation of CD4+ T lymphocytes in CLL patients receiving ibrutinib and rituximab front-line, thus free from the effects of previous chemotherapy.Examining the percentage and amount of Th1, Th2 and Th17 cytokine producing subsets in an analysis that followed the intra-individual variations, we documented that ibrutinib significantly shaped the T-cell profile, inducing an in vivo shift of the Th2/Th1 ratio, already evident after 14 days of in vivo ibrutinib treatment, mostly by limiting Th2 activation more than by expanding the Th1 compartment.Interestingly, the decrease of Th2/Th1 ratio over time was more prominent in the group of CLL patients with an unmutated IGHV status. The Th1 and Th2 cell subsets in patients treated with ibrutinib have been evaluated in other studies, with some inconsistent results -mostly depending on the variability of patients' selection and of experimental methods (i.e.plasmatic cell cytokines dosage vs flow cytometry) -and only partially comparable with our data (25-27).Yin et al. (26) examined 29 CLL patients under ibrutinib/ibrutinib+rituximab.They showed that the PB T-cell counts decreased and normalized under ibrutinib, in both the CD4 and CD8 subsets, at months +6 and +12 compared to the baseline.The serum concentration of a number of T-cell cytokines was measured and tended to decrease from month +3, remaining low thereafter.Interestingly, the IFNg/IL4 concentration ratio increased after 12 months of therapy, although not significantly, and this was suggested by the authors as a surrogate marker of the reported Th1 cell expansion upon ibrutinib (24). Niemann et al. (25) in 80 TN or relapsed/refractory (R/R) patients under ibrutinib showed a reduced expression of the exhaustion marker PD1 on T cells and a decrease of PB CD4+ and CD8+ T cells, more evident in patients with an unmutated or acalabrutinib (selective BTK inhibitor; n = 13) in second or further line of treatment.In contrast with the previous observations and in line with our results, ibrutinib (but not acalabrutinib) increased CD4+ and CD8+ T cells in CLL patients (more prominently in the effector/effector memory subsets), as a result of the diminished activation-induced cell death through ITK inhibition.Both agents induced a reduction in PD-1 and CTLA4 expression on T cells and of CD200 and BTLA expression, as well as of IL10 production on CLL cells, diminishing the immunosuppressive status induced by CLL cells.In contrast with our data, the authors were not able to document an in vivo change in T-cell polarization under ibrutinib; they showed no variation in frequency of activated CD4+ T cells producing IFNg, IL-4, TNFa or IL-2 from baseline to month +6, in contrast with our results. ITK plays an important role also in regulating signaling networks downstream of the TCR that govern the differentiation of Th17 cells and Th cells producing suppressive cytokines, including Foxp3+ Treg cells and Foxp3-Type 1 regulatory T (Tr1) cells (35,36).Recent works indicate that the absence of ITK impairs differentiation into Th17 and Tr1 cell subsets, but enhances the development of Tregs (35, 36), thus ibrutinib can likely modulate also the Th17/Tregs balance by direct inhibition of ITK (32). However, uncertain data are present in the literature with regard to Th17 and Treg cells in CLL patients under ibrutinib (32).In our paired sample analysis, we were able to detect a significant decrease in the frequency of Th17 cells after 18 months of ibrutinib treatment and in the Th17 cells absolute count from month +8 up to month +18.In line with our results, Niemann et al. (25) documented a 22% decrease of Th17, while Long et al. documented an increase in Th17 frequency after 6 months of ibrutinib (27).A recent study documented that Tregs decreased and returned to healthy levels in CLL patients receiving ibrutinib in first-line or for relapsed/refractory disease (28); in another, the number of Tregs remained unchanged, but the Tregs/CD4+ ratio was reduced under ibrutinib treatment but not with acalabrutinib (27), leaving this field open to further investigation. T-cell subsets could be also indirectly modulated by the reduction of the leukemic cell burden, besides the direct inhibition of ITK.Interestingly, in our study the decrease of the Th2/Th1 ratio was already induced after 14 days of in vivo ibrutinib treatment, well before the CLL cells start to decrease.Moreover, no linear correlation between the decrease of CLL lymphocytosis and the Th2/Th1 modulation was found by Pearson correlation analysis (data not shown).Analyzing the relationship between Th1 and Th2 modulation and disease activity, we noticed a correlation between a lower value of the Th2/Th1 ratio below 0.088 at month +8 and the achievement of a CR. It would be of interest to expand our observations to the different CLL compartments, such as the lymph nodes, in order to clarify the global impact of ibrutinib on T cells and the CLL microenvironment, and the role of lymphocyte mobilization from lymph nodes on the documented PB changes. Our data support the potential clinical use of ibrutinib beyond its anti-leukemic activity in CLL.Early evidences indicate that ibrutinib could be employed to control chronic graft-versus-host disease (37) and to enhance immunotherapy efficacy.Pre-clinical findings suggest that T-cell engaging CD19/CD3 bispecific antibodies against primary CLL cells from ibrutinib treated patients were more active than against samples from TN patients (38).The addition of ibrutinib to an anti-PD-L1 antibody was capable of enhancing tumor shrinkage in micebearing ibrutinib-resistant lymphoma, breast and colon cancer (39).Finally, ibrutinib enhanced ex vivo expansion, in vivo proliferation, and clinical activity of CAR T-cell therapy for CLL (40, 41). In conclusion, our results translate in a clinical setting the in vitro and in vivo pre-clinical evidences provided by Dubovsky et al. on the immunomodulatory role of ibrutinib (24).We suggest that in previously untreated patients, ibrutinib may restructure the host immune surveillance by an immune subversion of the Th2 dominant response, possibly generating a host anti-tumor immune activation, that may have an impact on patients' prognosis. The impact of ibrutinib immunomodulation on the infectious risk and disease control needs to be confirmed on a larger series of cases. AG designed the study and revised the data and the manuscript.RF critically revised the manuscript.All authors contributed to the article and approved the submitted version. ",
"section_name": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "The authors wish to thank the Associazione Italiana per la Ricerca sul Cancro (AIRC), Metastases Special Program, N°2 1198, Milan, Italy (RF); all the clinicians, the biologists and the patients involved in the trial. ",
"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 authors. ",
"section_name": "DATA AVAILABILITY STATEMENT",
"section_num": null
},
{
"section_content": "The studies involving human participants were reviewed and approved by the Ethic Committee of Azienda Policlinico Umberto I of Rome.The patients/participants provided their written informed consent to participate in this study. MCP, PM, and NP performed the experiments for Th1/Th2/ Th17.MCP wrote the manuscript.IDG performed the clinicobiologic correlations and contributed to write and revise the manuscript.MSDP performed flow cytometry for CLL analysis at baseline and subsequent time points.LVC contributed to data analysis and figures.LT, GR, AC, and SM enrolled and managed patients in the trial.AP and VA performed the statistical analysis.FRM wrote and run the clinical trial and enrolled patients. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2021.637186/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. ",
"section_name": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "The studies involving human participants were reviewed and approved by the Ethic Committee of Azienda Policlinico Umberto I of Rome.The patients/participants provided their written informed consent to participate in this study. ",
"section_name": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "MCP, PM, and NP performed the experiments for Th1/Th2/ Th17.MCP wrote the manuscript.IDG performed the clinicobiologic correlations and contributed to write and revise the manuscript.MSDP performed flow cytometry for CLL analysis at baseline and subsequent time points.LVC contributed to data analysis and figures.LT, GR, AC, and SM enrolled and managed patients in the trial.AP and VA performed the statistical analysis.FRM wrote and run the clinical trial and enrolled patients. ",
"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/fonc.2021.637186/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. ",
"section_name": "SUPPLEMENTARY MATERIAL",
"section_num": null
}
] |
10.1371/journal.pone.0228356
|
Clinical management and genomic profiling of pediatric low-grade gliomas in Saudi Arabia
|
Pediatric Low Grade Gliomas (PLGGs) display heterogeneity regarding morphology, genomic drivers and clinical outcomes. The treatment modality dictates the outcome and optimizing patient management can be challenging. In this study, we profiled a targeted panel of cancer-related genes in 37 Saudi Arabian patients with pLGGs to identify genetic abnormalities that can inform prognostic and therapeutic decision-making. We detected genetic alterations (GAs) in 97% (36/37) of cases, averaging 2.51 single nucleotide variations (SNVs) and 0.91 gene fusions per patient. The KIAA1549-BRAF fusion was the most common alteration (21/37 patients) followed by AFAP1-NTRK2 (2/37) and TBLXR-PI3KCA (2/37) fusions that were observed at much lower frequencies. The most frequently mutated) genes were NOTCH1-3 (7/37), ATM (4/37), RAD51C (3/37), RNF43 (3/37), SLX4 (3/37) and NF1 (3/37). Interestingly, we identified a GOPC-ROS1 fusion in an 8-year-old patient whose tumor lacked BRAF alterations and histologically classified as low grade glioma. The patient underwent gross total resection (GTR). The patient is currently disease free. To our knowledge this is the first report of GOPC-ROS1 fusion in PLGG. Taken together, we reveal the genetic characteristics of pLGG patients can enhance diagnostics and therapeutic decisions. In addition, we identified a GOPC-ROS1 fusion that may be a biomarker for pLGG.
|
[
{
"section_content": "Gliomas are common tumors in children and adolescents that display a broad range of clinical behaviors.[1] Most pediatric gliomas are benign and slow-growing lesions classified as grade I or II by the World Health Organization (WHO) classification criteria.[2][3][4][5][6][7][8] The most recent WHO classification in 2016 describes their histological features and provides a grading or malignancy scale.[6] Pediatric low-grade gliomas (pLGGs) account for approximately 35% of all childhood brain tumors and differ from adult low-grade gliomas (aLGGs) as they are seldom associated with IDH1/2 mutations, rarely undergo malignant transformation, and display high survival rates in response to traditional therapy.[9][10][11][12] The most common pLGG tumors in children are pilocytic astrocytoma (PA, Grade I) and diffuse astrocytoma (Grade II).[2][3][4][5] Other less common tumor types include pilomyxoid astrocytoma (Grade II), pleomorphic xanthoastrocytoma (PXA, Grade II), ganglioglioma (Grade I), angiocentric glioma (Grade I), subependymal giant cell astrocytoma (Grade I), and oligodendroglioma (Grade II).[6,13,14] pLGGs can be difficult to classify as they occur throughout the central nervous system (CNS) and often demonstrate overlapping microscopic features.[15,16] Historically, the cerebellum is the most common location with cerebellar LGGs accounting for 15% to 25% of all pediatric CNS tumors.These are followed by hemispheric (cerebral) gliomas (10%-15%), gliomas of the deep midline structures (10%-15%), optic pathway gliomas (5%), and brainstem gliomas (2%-4%). Surgery is the primary therapeutic modality for pilocytic astrocytomas and other LGGs.[17][18][19][20][21] Gross total resection (GTR) is often curative, despite the presence of residual microscopic disease.Clinical management strategies of children with subtotal resections are typically developed at a multidisciplinary pediatric neuro-oncology tumor board on a case-by-case basis.If the likelihood of functional impairment is minimal and neurosurgical intervention is deemed feasible, repeat surgery can often remove the residual tumor.In most cases, a \"wait and see approach\" is advocated, with follow-up brain MRI performed at 3 to 6 month intervals.Because pLGG tumors tend to be indolent by nature, the decision for repeat resection or adjuvant treatment can be postponed until measurable progression evidenced through neuroimaging or clinical symptoms are observed.[22][23][24][25] This interval may last several years and some tumors never progress.[13,26] When GTR is not possible, front-line chemotherapy regimens per the Children's Oncology Group Protocol A9952 are advocated.This involves a combination of carboplatin and vincristine that provides stable disease and tumor regression for an extended period.Chemotherapy also permits improved surgical resection of previously unresectable lesions.[27][28][29][30][31][32] Combined carboplatin and vincristine results in tumor reduction or stable disease and a 3-year PFS of 68%.[2,27,[33][34][35] Radiotherapy is typically contraindicated in children, particularly those with NF1 germline mutations, PAs, and other LGGs, including cases of diencephalic and optic pathway tumors.Even highly focused radiation therapy at these locations does not eliminate the associated cognitive, endocrine, or vascular risks.[33,36,37] In this study, we sought to illustrate how correlating genetic alterations with histologic and clinical features can improve pLGG classification and treatment decisions for patients in Saudi Arabia (SA).Our cohort included cases from a tertiary care center in King Fahad Medical City (KFMC), which is a primary referral center for pediatric neoplasms and King Khalid University Hospital (KKUH) in SA and reflects the distribution of pLGG subtypes across the kingdom. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "This retrospective study was performed with IRB#16-310 following the relevant ethical guidelines and regulations from the King Fahad Medical City, Riyadh, KSA.King Fahad Medical City's International Review Board reviewed and approved this study before the study began and waived the requirement for informed consent for the archival samples.The study was performed on 37 children where tissue was available (age <16 years) who were newly diagnosed with pathologically confirmed pLGG between January 2011 and January 2017.We reviewed the molecular, clinical and therapeutic aspects and treatment outcomes of the pLGG patients in KFMC (S1 Fig) .We collected essential demographic and disease-specific characteristics and radiology images to assess the extent of tumor resection.Information on neurosurgical management was obtained from operative records and standardized neurosurgical reports.All data were fully anonymized before we accessed them.Archived pathology specimens were reviewed by a board-certified neuropathologist (MA).Progression-free survival (PFS) was defined as the minimum time to tumor progression, second malignancy, or death from any cause. ",
"section_name": "Patient cohort",
"section_num": null
},
{
"section_content": "Next-generation sequencing (NGS) was performed using the Oncomine Comprehensive Assay v3 system, a targeted assay that enables the detection of relevant SNVs, CNVs, gene fusions, and indels from 161 genes (S2 Fig) .Multiplex DNA primers were used to prepare amplicon libraries from formalin-fixed paraffin-embedded (FFPE) tumor samples.Assays were performed using the Ion S5 System and Ion 540 Chip (Thermofisher Scientific, USA). ",
"section_name": "Next-generation sequencing",
"section_num": null
},
{
"section_content": "Overall survival (OS) was measured from the date of diagnosis until the date of death from any cause or date of last contact.PFS and OS were estimated using the Kaplan-Meier method.A Pvalue < 0.05 was considered statistically significant.A secure electronic database was created for storage and data analysis.The data were entered and analyzed using SPSS statistical package version 23. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Thirty-seven patients were assessed (19 males, 18 females) with a median age at diagnosis of 1-12 years (range: 12-154 months) with histologically proven low-grade astrocytoma (Grades I, II) (Fig 1A).Most tumors (31/37 cases, 83.7%) were classified as pilocytic astrocytomas.Four patients were classified with pleomorphic xanthoastrocytoma (10.8%) and two patients had diffuse gliomas (5.4%), (Fig 1B). Cerebellar tumors were encountered in 18/37 patients (48.6%) accounted for 8/37 tumors (21.6%).Cerebral hemisphere/cortex tumors also occurred in 8/37 patients (21.6%), the second most common sites.A total of 4/37 patients had hypothalamic tumors with optic pathway involvement, 3/37 patients (8.10%) had suprasellar masses, 2/37 (5.4%) patients had spinal cord tumors as the primary site, and 2 (5.40%) patients had brain stem tumors (S1 Table andFig 1C). Amongst the patients, 26/37 (70.3%) had initial surgery followed by observational serial MRI (Fig 2A) and 9 of these patients experienced relapse/progression (Fig 2B).In total, 10 patients received adjuvant chemotherapy after surgery, two of which had relapse/progression after first-line chemotherapy, with a single patient relapsing on two occasions (S1 Fig) .A single patient (1/37) received adjuvant radiotherapy due to incomplete surgical resection of the tumor and showed no relapse (Fig 2A).Amongst the surgical procedures employed, 10/26 underwent complete surgical excision, 7/26 underwent partial excision, 2/37 had subtotal resection, and a single patient underwent biopsy.The surgical procedures for the remaining patients were not defined.All the patients are still alive, whilst 11 (29.7%)experienced relapse/ progression.The median progression-free survival time was 36.5 months. ",
"section_name": "Cohort demographics and clinical management",
"section_num": null
},
{
"section_content": "Gene alterations were identified in 36/37 (97.3%) of pLGGs, averaging 2.51 single nucleotide variations and 0.91 gene fusions per patient (Fig 3A).The KIAA1549-BRAF fusion was most common (21/37 patients) followed by AFAP1-NTRK2 (2/37) and TBLXR-PI3KCA (2/37) ).Two of these patients harbored both ESR1-CCDC170 and KIAA1549-BRAF fusions, whilst a single case co-harbored AGK-BRAF and KIAA1549-BRAF fusions.One case harbored TBL1XR1-PIK3CA, EIF3E-RSPO2 fusions (S2 Table ).Regarding anatomic location and BRAF fusions, most were located in cerebellum/ posterior fossa (S1 and S2 Tables).Of the relapse cases, 10/12 harbored KIAA1549-BRAF fusions.Of the cases lacking KIAA1549-BRAF fusions, a patient harboring a GOPC-ROS1 fusion of was of interest, as this alteration has been previously reported in an undefined glioblastoma patient.The clinical outcome of this case was not previously reported in the literature.[38,39] ",
"section_name": "Genetic alterations in pLGGs",
"section_num": null
},
{
"section_content": "Golgi-associated PDZ and coiled-coil motif-containing (GOPC) protein regulates the intracellular trafficking of membrane proteins.[40] The ROS proto-oncogene 1 is a receptor tyrosine kinase expressed in lung and brain tissues.[41] GOPC-ROS1 fusions have been identified in a \"not otherwise specified\" (NOS) single case of glioblastoma (deemed to be neither pLGG nor pHGG) in which the tumor also harbored mutations in other glioma-associated genes, including TP53 and PTPN11, [38] and in one case of pHGG.[39] In the GOPC-ROS1 fusion pHGG case, the patient underwent gross total resection, at 4 years of age, followed by adjuvant highdose chemotherapy and autologous hematopoietic stem cell rescue.At 30 months post-transplantation, the patient remained disease-free.[39] We detected a likely pathogenic GOPC-ROS1 fusion in a pLGG patient who also harbored a RAD15C variant of uncertain significance.He is a previously healthy 8-year-old boy who presented with unprovoked recurrent convulsions.MRI imaging revealed a left parietal mass measuring (6 × 5 cm) with a mass effect and vasogenic edema (Fig 4).The patient underwent GTR.The patient is currently disease free and showed excellent postoperative recovery with no neurological deficits or evidence of progression (S1 Table ).Histopathological examination revealed a low-grade glioma with low-to-moderate cellularity and a biphasic growth pattern comprising tumor cells with piloid and oligodendroglial morphologies and associated with Rosenthal fibers ( ).Currently, the patient is stable with no symptoms or signs suggestive of tumor recurrence, managed with stable serial follow-up MRI. ",
"section_name": "Identification of the GOPC-ROS1 fusion in a single LGG patient",
"section_num": null
},
{
"section_content": "In this study, we reviewed the clinical management and performed a targeted genetic screening of a panel of cancer-related genes in 37 pediatric LGGs.Amongst the pLGGs, the most common alterations were KIAA1549-BRAF fusions (26/37).From histological analysis, most patients were diagnosed with pilocytic astrocytomas (31/37).This was comparable to previous findings in which 60% to 80% of PAs harbored BRAF fusions.[42] BRAF V600E mutations were observed in only 2/37 patients, while H3F3A (K27M) histone mutations were not detected.Tumors were predominantly located in the cerebellum/posterior fossa (18/37) and cerebral hemisphere/cortex (8/37).The majority of tumors harboring BRAF fusions were located in the cerebellum/posterior fossa (Fig 1C, S2 Table ), which was consistent with the association between tumor location and BRAF fusions reported in other cohorts.[42,43] Tumors in the cerebellum are traditionally amenable to surgical intervention, with resection rates of ~94% and an overall tumor recurrence rate of ~19%.[42] We observed higher rates of recurrence (12 of 37 patients, 32.43%), raising the possibility that BRAF fusions may not be directly associated with an improved outcome in SA pLGGs, as reported in previous studies.[43,44] Because KIAA1549-BRAF is rarely detected in pHGGs, including anaplastic astrocytoma and glioblastoma, BRAF fusions can genetically distinguish pLGG from pHGG in the Saudi cohort.We suggest that in the future, the identification of BRAF fusions can guide patient treatment as targeted molecular therapies are discovered.It is important that a diagnostic test for these fusions is readily available in SA in a clinical setting.).Notch signaling is evolutionarily conserved and known to regulate cell proliferation, apoptosis, migration, and differentiation.In mammals, Notch signaling is composed of Notch1-4 receptors and Dll1-3-4, Jagged1-2 ligands that develop and maintain the CNS.The frequency of these mutations is perhaps surprising since higher expression of ASCL1, Dll1, Notch1, -3, -4 have been shown to correlate with a higher glioma grade and poorer prognosis, implicating Notch signaling in more undifferentiated and aggressive tumor phenotypes.We observed no association between Notch mutants and relapse/disease progression in our cohort, indeed a patient with co-occurring Notch2/3 mutations did not relapse following surgical intervention (S2 Table ). Among the less frequent mutations, we observed alterations in RAD51C, a component of the DNA double-strand repair pathway, the E3 ubiquitin ligase RNF43, and the central checkpoint gene ATM that is involved in the repair of DNA damage after ionizing irradiation to be associated with the risk of brain tumors.[45] Mutations in DNA repair pathways are typically associated with therapeutic resistance and chemotherapy-induced mutagenesis.[46][47][48] This highlights the importance of genetic assessment following surgical resection.Should RAD51Cand RNF43-mutated pLGGs recur and undergo malignant progression to a higher histological grade, postoperative adjuvant treatment using immunotherapy approaches and checkpoint inhibitors may be employed as opposed to chemotherapeutic interventions.Regarding RNF43, mutations affecting this gene were loss-of function mutations, likely leading to the activation of pro-oncogenic Wnt signaling by interfering with the RNF43-mediated ubiquitination of the frizzled receptor.[49] Concurrent inhibition of WNT signaling components may therefore benefit these pLGG patients.In this regard, over 25 antibodies, 53 polypeptides/proteins and 21 chemicals are currently available to inhibit WNT signaling, some of which are clinically approved.[50,51] Regarding our case report, the identification of the GOPC-ROS1 fusion provides insight into disease pathophysiology and the use of the FDA-approved ROS1 inhibitor crizotinib in SA pLGG because this was identified as a gain-of-function mutation in the Oncomine database (S2 Fig) .Previous studies have also highlighted the GOPC-ROS1 fusion as a resistance marker to chemotherapy in lung cancer, indicating that this fusion may be prognostic for a poor chemotherapeutic outcome.[41] As this patient achieved a gross total resection, there was no indication for adjuvant chemotherapy or radiation therapy.To date, the patient is stable with no symptoms or signs suggestive of tumor recurrence.Given the approval of crizotinib to treat late-stage non-small cell lung cancers that are ROS1 positive, [41,52] this may represent a potential treatment option should this patient experience disease recurrence. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Interrogation of the sequencing data in the SA cohort has revealed BRAF fusions as critical biomarkers to predict resectable pLGG.We have further identified that RAD51C, RNF43, and ATM may hold prognostic value in the SA population.We identified a rare GOPC-ROS1 fusion in pLGG patients lacking BRAF alterations, which may represent a genomically-distinct subgroup of pLGGs that could be targeted with crizotinib.To our knowledge this is the first report of this fusion in pLGG.These findings demonstrate how genetic profiling can guide optimal treatment strategies for pLGG in the Saudi population. ",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "The authors thanks King Abdulaziz City for Science and Technology and the Saudi Human Genome Project for technical support.This Study was funded by KFMC-IRF 17-65 (MA) and Sanad Cancer research foundation RGP 2017-1 (MA) ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "This Study was funded by KFMC-IRF 17-65 (MA) and Sanad Cancer research foundation RGP 2017-1 (MA).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": "Formal analysis: Latifa AlMubarak, Rasha Alaljelaify, Mariam AlSaeed, Amal Almutairi, Fatmah Alqubaishi, Malak Abedalthagafi. Investigation: Nahla A. Mobark, Musa Alharbi, Malak Abedalthagafi. Methodology: Nahla A. Mobark, Musa Alharbi, Malak Abedalthagafi. Project administration: Malak Abedalthagafi. Resources: Malak Abedalthagafi. Supervision: Malak Abedalthagafi. Validation: Malak Abedalthagafi. Writing -original draft: Lamees Alhabeeb, Malak Abedalthagafi. Writing -review & editing: Malak Abedalthagafi. ",
"section_name": "Supporting information",
"section_num": null
},
{
"section_content": "Formal analysis: Latifa AlMubarak, Rasha Alaljelaify, Mariam AlSaeed, Amal Almutairi, Fatmah Alqubaishi, Malak Abedalthagafi. ",
"section_name": "Supporting information",
"section_num": null
},
{
"section_content": "Investigation: Nahla A. Mobark, Musa Alharbi, Malak Abedalthagafi. Methodology: Nahla A. Mobark, Musa Alharbi, Malak Abedalthagafi. Project administration: Malak Abedalthagafi. Resources: Malak Abedalthagafi. Supervision: Malak Abedalthagafi. Validation: Malak Abedalthagafi. Writing -original draft: Lamees Alhabeeb, Malak Abedalthagafi. Writing -review & editing: Malak Abedalthagafi. ",
"section_name": "Funding acquisition: Malak Abedalthagafi.",
"section_num": null
}
] |
10.3389/fonc.2021.571476
|
Beta-Elemene Reduces the Malignancy of Non-Small Cell Lung Cancer by Enhancing C3orf21 Expression
|
<jats:sec><jats:title>Background</jats:title><jats:p>Beta-elemene has potent anti-tumor effect, but its anti-tumor mechanism remains unclear. Chromosome 3 open reading frame 21 (C3orf21) acts as a tumor suppressor. This study tested whether the anti-tumor effect of beta-elemene was associated with modulating C3orf21 expression in non-small cell lung cancer (NSCLC).</jats:p></jats:sec><jats:sec><jats:title>Materials and Methods</jats:title><jats:p>The impact of beta-elemene on C3orf21 expression in NSCLC cells was quantified. The stable C3orf21 silencing A549 and over-expressing PC-9 cells were established and their effects on the beta-elemene-attenuated proliferation, wound healing and invasion of NSCLC cells as well as the expression of key regulators and signal events were determined.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Beta-elemene significantly up-regulated C3orf21 expression in NSCLC cells. Beta-elemene treatment significantly attenuated the proliferation, wound healing and invasion of NSCLC cells, which were significantly mitigated by C3orf21 silencing, but enhanced by C3orf21 over-expression. Similar patterns of beta-elemene-modulated cyclinD1, c-Myc, COX2, MMP2, MMP9, VEGF, PTEN and Notch1 expression were detected in NSCLC cells.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>Such data indicated that beta-elemene treatment attenuated the malignancy of NSCLC cells by up-regulating C3orf21 expression. Our findings may provide new mechanisms underlying the pharmacological action of beta-elemene.</jats:p></jats:sec>
|
[
{
"section_content": "Non-small cell lung cancer (NSCLC) is the most prevalent malignant lung tumors with high cancerrelated mortality worldwide (1,2).At present, the major treatments for NSCLC are surgical resection, target therapies and chemotherapy.Although these therapeutic strategies for NSCLC have greatly prolonged the survival of patients, many NSCLC patients commonly develop resistance to target therapies (3).This, together with the fact that many NSCLC patients are diagnosed at advanced stages, leads to a low rate of five-year survival (4).Hence, discovery of new therapeutic reagents and their acting mechanisms will be of high significance in management of NSCLC patients. Beta-elemene, a natural lipid-soluble plant drug, can be extracted from traditional Chinese medicine Rhizoma zedoariaem (5,6).Previous studies have shown that betaelemene has potent anti-tumor activity in several types of malignant tumors (7)(8)(9)(10).More importantly, beta-elemene is relatively safe and has been widely used as an effective anticancer drug in humans (6,11).Recent studies have shown that betaelemene can reduce the malignancy of NSCLC cells by inhibiting their proliferation, migration and invasion (5,8).Furthermore, beta-elemene can sensitize glioblastoma multiforme cells to gefitinib (12) and synergistically enhances its therapeutic effect on inhibiting stemness and progression of lung cancer by downregulating EZH2 expression (13).However, the exact molecular mechanisms underlying the therapeutic action of beta-elemene remain largely indistinct, which may be an important obstacle for improving and refining the clinical application of beta-elemene. Our previous study has indicated that chromosome 3 open reading frame 21 (C3orf21) can regulate the development of lung adenocarcinoma (14,15).In this study, we tested the hypothesis that beta-elemene could modulate C3orf21 expression to attenuate the malignancy of NSCLC cells.The results indicated that beta-elemene treatment reduced the proliferation, wound healing and invasion of NSCLC cells by increasing C3orf21 expression.Such findings may provide new pharmacological evidence to explain why beta-elemene inhibits the malignancy of NSCLC. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "Beta-elemene [1-methyl-1-vinyl-2,4-diisopropenyl-cyclohexane, C15H24, molecular weight of 204.35, Figure 1 (16)] is a sesquiterpene compound extracted from the dried rhizome of Rhizoma curcumae.The beta-elemene injection (xx% of betaelemene, National Medical Product Administration approval Number: Chinese medicine H10960114) was produced by Dalian Huali Jingang Pharmaceutical (Dalian, China). ",
"section_name": "Beta-Elemene",
"section_num": null
},
{
"section_content": "We searched the potential targets of beta-elemene using the BATMAN-TCM (http://bionet.ncpsb.org/batman-tcm/)database and STITCH (http://http://stitch.embl.de)database to predict the beta-elemene-associated genes and proteins.The BATMAN-TCM is an online bioinformatics analysis tool specially designed for studying the molecular mechanisms of traditional Chinese medicine, and is based on traditional Chinese medicine ingredients' target prediction (17).The STITCH also is online Search Tool for Interacting Chemicals, which integrates these disparate data sources for 430,000 chemicals into a single, easy-to-use resource.The user can get a quick overview of the potential effects of the chemical on its interaction partners by STITCH database (18). ",
"section_name": "Bioinformatic Analysis of Beta-Elemene-Associated Genes and Proteins",
"section_num": null
},
{
"section_content": "We obtained human NSCLC A549, PC-9, NCI-H1975, MSTO-211H and NCI-H226 cells from Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences.We identified them by STR.We grew them in RMPI1640 medium (Invitrogen) containing 10% fetal bovine serum (FBS, Gibco) in at 37°C 5% CO 2 .We treated the cells with, or without, the indicated concentrations of beta-elemene (Dalian Huali Jingang Pharmaceutical, Dalian, China) for varying time periods. ",
"section_name": "Cells",
"section_num": null
},
{
"section_content": "We cloned the DNA fragments for the expression of control and specific shRNAs (Table 1, Sangon Biotech, Shanghai, China) or the cDNA for C3orf21 expression into pLKO-ZSG-Puro, together with pCMV△R8.92 and pVSVG-I, to generate different types of lentiviruses in 293T cells.Subsequently, we transduced A549 cells with control or lentivirus for the expression of C3orf21-specific shRNA (Addgene) at multiplicity of infection of 4 in the presence of 8 mg/ml hexadimethrine bromide (Sigma).Similarly, we transduced PC-9 cells with control or lentivirus for the over-expression of C3orf21.Three days later, we treated the cells with puromycin (500 ng/ml) or blasticidin (10 mg/ml) to generate shCon, sh-C3orf21 stably silencing A549 and control, C3orf21 over-expressing PC-9 cells, respectively.We tested the efficacy of C3orf21 silencing or over-expression by fluorescent microscopy and Western blot assays. ",
"section_name": "Lentivirus Transduction",
"section_num": null
},
{
"section_content": "We extracted total RNA from the different groups of cells using TRIzol (Invitrogen) and after qualification and quantification using an Agilent Bioanalyzer 2100 (Agilent Technologies), we reversely transcribed RNA samples into cDNAs using an iScript cDNA Synthesis Kit (BIO-RAD).Subsequently, we quantified the relative levels of mRNA transcripts by qRT-PCR using SYBR Premix ExTaqTM II kit (TaKaRa) and specific primers (Table 1).We performed RT-PCR in triplicate at 94°C for 10 s, and 40 cycles of 94°C for 5 s, 52°C for 30 s, 72°C for 15 s.We analyzed the data using the 2 -DDCt method. ",
"section_name": "Quantitative Real-Time-PCR (qRT-PCR)",
"section_num": null
},
{
"section_content": "We determined the impact of beta-elemene on the proliferation of NSCLC cells by CCK-8 assay (Beyotime Shanghai China).Briefly, the cells (2-3.5 × 10 3 /well) from each group were grown in 96-well plates and treated in triplicate with beta-elemene at different concentrations for 24 h.Their proliferation was quantified with 10 ml/well of CCK-8 at 450 nm. ",
"section_name": "CCK-8 Cell Assay",
"section_num": null
},
{
"section_content": "The different groups of cells (2 × 10 5 cells/well) were cultured in 6-well plate up to 80-90% confluence.The cell monolayer was wounded with a sterile micropipet tip and treated with, or without, the indicated doses of beta-elemene in serum-free medium.The monolayer of cells was photoimaged before and 24 h after beta-elemene treatment under an Olympus CKX-41 inverted microscope.The migrated areas of cancer cells were measured by Image J software. ",
"section_name": "Wound Healing Assay",
"section_num": null
},
{
"section_content": "We examined whether beta-elemene could modulate NSCLC cell invasion by transwell invasion assay using the BioCoat Matrigel Invasion Chamber (Corning), according to the manufacturer's recommended instructions.In brief, the different groups of cells (4 × 10 3 cells/well) were cultured in triplicate in the upper chamber that had been coated with Matrigel (REF 3422, Corning).The bottom chambers were filled with 10% FBS medium.After 48-h culture, the invaded cells on the bottom surface of the upper chamber membranes were fixed and stained with crystal violet, followed by photoimaged.The invaded cells in five fields of each membrane were counted. ",
"section_name": "Transwell Invasion Assay",
"section_num": null
},
{
"section_content": "We quantified the relative levels of interested proteins to the control GAPDH by Western blot.Briefly, we harvested each group of cells and lysed them in cell lysis buffer (Beyotime) containing a cocktail of protease inhibitors (Rocha).After qualification and quantification, we analyzed the cell lysates (30 µg/lane) by SDS-PAGE, and transferred to polyvinyl dene fluoride membranes (Millipore).After blocked, we incubated the membranes with anti-C3orf21 (20913-1-AP, Proteintech), anti-Notch1 (68309T), anti-GAPDH (2118S, Cell Signaling Technology) and detected the bound antibodies with HRPconjugated second antibodies, followed visualizing using the enhanced chemiluminescence (Pierce, Rockford, USA).We quantified the data using Image J. ",
"section_name": "Western Blot",
"section_num": null
},
{
"section_content": "Data are present as mean ± SD.The difference between two groups was analyzed by Student's T test, and the difference among multiple groups was analyzed by Two-way ANOVA using the GraphPad Prism 5.0 software.Statistical significance was defined when a P-value of ≤0.05. ",
"section_name": "Statistical Analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "To search the potential genes and proteins associated with betaelememe, we performed bioinformatics analysis using betaelemene (6918391, PubChem CID) as a key word to search the BATMAN-TCM database.With a cutoff score ≥5, we obtained 522 genes associated with beta-elemene (Figure 2).To further search the beta-elemene-associated proteins, we used the STITCH database and found that fifty proteins were associated with beta-elemene, including XXYLT1, Notch and others (Figure 3). ",
"section_name": "Bioinformatic Analysis of Beta-Elemene Targeting Genes and Proteins",
"section_num": null
},
{
"section_content": "C3orf21 has shown to act as a tumor suppressor of several types of malignant tumors.To understand the antitumor effect of betaelemene, we tested whether beta-elemene treatment could ",
"section_name": "Beta-Elemene Enhances C3orf21 Expression in NSCLC Cells",
"section_num": null
},
{
"section_content": "Sequences (5'-3') Application modulate C3orf21 expression in several NSCLC cell lines. Western blot analysis indicated that C3orf21 expression varied in the different NSCLC cell lines (Figure 4A) and treatment with beta-elemene (10 mg/ml) for 24 h significantly up-regulated C3orf21 expression in these NSCLC cells (p < 0.05-p < 0.001, Figure 4B).Given that A549 cells expressed relatively higher levels of C3orf21 while PC-9 expressed lower levels of C3orf21, we generated C3orf21 stably silenced A549 and C3orf21 overexpressed PC-9 cells (Figures 4C,D) and these cell lines were valuable for determining the role of altered C3orf21 expression in regulating the beta-elemene-mediated anti-NSCLC effects. ",
"section_name": "Primer",
"section_num": null
},
{
"section_content": "To investigate how altered C3orf21 expression modulated the anti-NSCLC effect of beta-elemene, we first tested the impact of beta-elemene on A549 and PC-9 cell viability by CCK-8 assays.We found that treatment with beta-elemene significantly decreased A549 and PC-9 cell viability in a dose-dependent manner (Figures 5A,B).Furthermore, C3orf21 silencing dramatically mitigated the inhibitory effects of beta-elemene on the proliferation of A549 cells while C3orf21 over-expression remarkably enhanced its inhibitory effects on the proliferation of PC-9 cells (Figures 5C,D).These data indicated that betaelemene attenuated the proliferation of NSCLC cells, which was regulated by C3orf21 expression. ",
"section_name": "Beta-Elemene Inhibits the Proliferation of NSCLC Cells, Dependent on C3orf21 Expression",
"section_num": null
},
{
"section_content": "To further explore how beta-elemene modulated the malignancy of NSCLC cells, we tested whether altered C3orf21 expression could change the effect of beta-elemene on the wound healing of shNC-A549, sh-C3orf21-A549, control-PC-9 and C3orf21-PC-9 cells.While C3orf21 silencing significantly increased the wound healing of shNC-A549 cells treatment with beta-elemene significantly decreased their wound healing and the inhibitory effects tended to be dose-dependent (Figure 6A).Similarly, betaelemene treatment also significantly attenuated the wound healing of C3orf21-silenced A549 cells, but the wound healing effects in the C3orf21-silenced A549 cells were significantly stronger relative to their corresponding shNC-A549 cells.Furthermore, beta-elemene treatment also significantly minimized the wound healing of control-PC-9 cells and further decreased it in the C3orf21-over-expressed PC-9 cells (Figure 6B).A similar pattern of beta-elemene treatment on the invasion of shNC-A549, sh-C3orf21-A549, control-PC-9 and C3orf21-PC-9 cells was observed by transwell invasion assays (Figures 6C,D).Collectively, C3orf21 silencing mitigated the inhibition of beta-elemene on the wound healing and invasion of NSCLC while C3orf21 over-expression enhanced its inhibitory effects in NSCLC cells. ",
"section_name": "Beta-Elemene Attenuates the Wound Healing of NSCLC Cells Dependent on C3orf21 Expression",
"section_num": null
},
{
"section_content": "To further understand the pharmacological action of betaelemene in attenuating the malignancy of NSCLC cells, we examined how beta-elemene treatment could modulate the levels of Cyclin D1, c-Myc, COX2, MMP2, MMP9, VEGF and PTEN as well as Notch1 mRNA transcripts and protein expression in different groups of NSCLC cells.As shown in Figure 7, C3orf21 silencing significantly increased the relative levels of cyclin D1, c-Myc, Cox2, MMP2, MMP9, VEGF, but decreased PTEN mRNA transcripts in A549 cells.Conversely, C3orf21 over-expression had opposite effects on their expression in PC-9 cells, relative to their controls.Treatment with betaelemene significantly decreased Cyclin D1, c-Myc, COX2, MMP2, MMP9, PTEN, but increased VEGF mRNA transcripts in both control and C3orf21-silenced A549 cells, relative to that of untreated cells.In addition, beta-elemene treatment also deceased Cyclin D1, c-Myc, COX2, MMP2, MMP9 and VEGF, but increased PTEN mRNA transcripts in both control and C3orf21-over-expressed PC-9 cells, compared with that in their untreated cells.Western blot analysis indicated that C3orf21 silencing up-regulated Notch1 expression in A549 cells while C3orf21 over-expression down-regulated Notch1 expression in PC-9 cells (Figure 8).Treatment with beta-elemene significantly reduced Notch1 expression in all groups of cells, particularly in the C3orf21-over-expressed PC-9 cells.Together, such data indicated that beta-elemene treatment modulated the expression of several key regulators and signal events in NSCLC cells, dependent on C3orf21 expression. ",
"section_name": "Beta-Elemene Modulates the Expression of Several Regulators and Signal Events in NSCLC Cells, Dependent on C3orf21 Expression",
"section_num": null
},
{
"section_content": "NSCLC is the most prevalent malignancy, and current treatments for NSCLC include surgical resection, target therapies and chemotherapy, which have side effects.Hence, discovery of new safe and therapeutic reagents is urgently needed.Previous studies have shown that beta-elemene has potent antitumor activity and can minimize the drug resistance of some types of tumor cells (19)(20)(21).In this study, we found that beta-elemene significantly enhanced C3orf21 expression in NSCLC cells.Beta-elemene treatment dramatically decreased the proliferation, would healing and invasion of NSCLC cells, which were mitigated by C3orf21 silencing, but enhanced by C3orf21 over-expression in vitro.The significantly decreased proliferation, wound healing and invasion of NSCLC cells indicated that beta-elemene remarkably attenuated the malignancy of NSCLC cells by up-regulating C3orf21 expression.Such novel findings extended previous observations and support the notion that C3orf21 acts as a suppressor in inhibiting the development and progression of malignant tumors.Moreover, C3orf21 over-expression further enhanced the therapeutic effect of beta-elemene, which suggests that C3orf21 may be a new therapeutic target for intervention of NSCLC. To understand the pharmacological action of beta-elemene, we tested whether beta-elemene treatment could modulate the expression of key regulators for cell behaviors and signal events in NSCLC cells.We found that C3orf21 silencing remarkably increased the relative levels of Cyclin D1, c-Myc, Cox2, MMP2, MMP9, VEGF, besides Notch1, but decreased PTEN expression while C3orf21 over-expression displayed opposite effects in NSCLC cells.Such results extended previous observations that C3orf21 inhibits the Notch signaling (22).It is well known that Cyclin D1, c-Myc and Cox2 can promote the proliferation of tumor cells (23) while PTEN can suppress the PI3K/AKT/mTOR signaling (24).Furthermore, VEGF is an important angiogenic factor and MMP2/9 are crucial for tumor cell motility (25).In addition, decreased levels of C3orf21 expression is related to a poor prognosis in patients with lung cancer (26).Functionally, C3orf21 can prolong the O-linked xylose-glucose (14).The inhibitory effect of C3orf21 on the Notch signaling is likely associated with the differential O-linked glycosylation of its extracellular domain (NECD) (27,28).More importantly, we found that beta-elemene treatment significantly decreased cyclin D1, c-Myc, Cox2, MMP2/9 expression, which were attenuated by C3orf21 silencing and enhanced by C3orf21 over-expression in NSCLC cells.Interestingly, beta-elemene treatment significantly increased VEGF expression, but decreased PTEN expression in C3orf21-sielnced A549 cells.The same treatment had opposite effects on the VEGF and PTEN expression in the C3orf21-overexpressed PC-9 cells.It is possible that C3orf21 may through the similar mechanisms, regulate the expression of other enhanced by C3orf21 over-expression.Similarly, we found that beta-elemene treatment significantly altered the expression of key regulators of malignant behaviors and signal events, which were modulated by altered C3orf21 expression in NSCLC cells.Thus, our findings may provide new insights into the pharmacological action of beta-elemene in inhibiting the development and progression of NSCLC. ",
"section_name": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "This study was supported by grants from the Zhejiang Provincial Medical and Health Scientific and Technical Foundation (No.2017171306, 2018KY296 and 2020KY470). ",
"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": "HC and YZ were responsible for study design, performing experiments, statistical analysis, and manuscript writing.LR and YW collected the data and 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. ",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "HC and YZ were responsible for study design, performing experiments, statistical analysis, and manuscript writing.LR and YW collected the data and 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
}
] |
10.1038/s41467-017-01500-9
|
Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma
|
<jats:title>Abstract</jats:title><jats:p>Notch1 transactivates <jats:italic>Notch3</jats:italic> to drive terminal differentiation in stratified squamous epithelia. Notch1 and other Notch receptor paralogs cooperate to act as a tumor suppressor in squamous cell carcinomas (SCCs). However, Notch1 can be stochastically activated to promote carcinogenesis in murine models of SCC. Activated form of Notch1 promotes xenograft tumor growth when expressed ectopically. Here, we demonstrate that Notch1 activation and epithelial–mesenchymal transition (EMT) are coupled to promote SCC tumor initiation in concert with transforming growth factor (TGF)-β present in the tumor microenvironment. We find that TGFβ activates the transcription factor ZEB1 to repress <jats:italic>Notch3</jats:italic>, thereby limiting terminal differentiation. Concurrently, TGFβ drives Notch1-mediated EMT to generate tumor initiating cells characterized by high CD44 expression. Moreover, Notch1 is activated in a small subset of SCC cells at the invasive tumor front and predicts for poor prognosis of esophageal SCC, shedding light upon the tumor promoting oncogenic aspect of Notch1 in SCC.</jats:p>
|
[
{
"section_content": "N otch signaling regulates cell fate in a context-dependent manner 1 .The ligand-activated intracellular domain of Notch (ICN) forms a transcriptional activation complex with the transcription factor CSL and the co-activator MAML.Notch1 drives terminal differentiation in stratified squamous epithelia 2,3 in concert with other Notch receptor paralogs 4,5 .Histopathology of squamous cell carcinomas (SCCs) features squamous-cell differentiation, a process normally regulated via direct transcriptional activation of Notch3 by ICN1, the activated form of Notch1, in esophageal epithelia 4 .Loss-of-function Notch1 mutations are found in SCCs 6,7 , suggesting a tumor suppressor role for Notch1 5,8,9 .However, Notch1 can be stochastically activated or inactivated, with either scenario resulting in promotion of carcinogenesis in murine models of SCC 10 .Many human SCC cell lines express ICN1 and ectopic ICN1 expression promotes xenograft tumor growth 11,12 .While pharmacological modulation of Notch paralogs represents an attractive strategy for cancer therapy 13 , a more detailed understanding of the functional role of the Notch pathway as it relates to tissue biology in the context of health and disease is necessary to guide such approaches. In addition to squamous-cell differentiation, Notch1 regulates cell cycle 3,12,14 , senescence 12 , and epithelial-mesenchymal transition (EMT) [15][16][17] .Acquisition of mesenchymal properties facilitates malignant transformation by limiting oncogeneinduced senescence 18,19 .In human esophageal squamous cell carcinoma (ESCC), the deadliest form of all human SCCs 20 , EMT is associated with chemoresistance and poor prognosis [21][22][23] .EMT also regulates cancer stem cells (CSCs) 24,25 .CSCs defined by high CD44 expression (CD44H) have been identified in various tumor types, including SCCs [26][27][28][29] .In transformed esophageal and oral keratinocytes, cells with low CD44 expression (CD44L) and epithelial properties are converted to CD44H cells with mesenchymal traits in response to transforming growth factor (TGF)-β 30,31 , a potent EMT inducer present in the tumor microenvironment 32 .During TGFβ-mediated EMT, expression of the Notch ligand JAG1 is induced via ZEB1 12,15,33 , a transcription factor essential in TGFβ-induced EMT 34,35 and microRNA-mediated regulation of Notch signaling 33,36,37 .While emerging lines of evidence support Notch1 as a positive effector of EMT 15-17, 37, 38 , Notch3 limits the expansion of EMTcompetent esophageal keratinocytes 11 .Thus, although Notch1 and Notch3 cooperate to drive squamous-cell differentiation 4 , these Notch paralogs may play opposing roles in EMT and, potentially, regulation of CSC dynamics.The precise molecular mechanisms through which Notch signaling regulates distinct cell fates in a context-dependent manner have yet to fully elucidated. Here, we aimed to define the functional role of Notch1 in SCC.We demonstrate that Notch1 activation and EMT are coupled to promote tumor initiation and intratumoral cancer cell heterogeneity in SCC.We find that the transcription factor ZEB1 represses NOTCH3, thereby limiting ICN1-induced differentiation while permitting ICN1-mediated EMT.Moreover, ICN1 expression in a small subset of SCC cells at the invasive tumor front predicts independently for poor prognosis of ESCC.These findings suggest an oncogenic role for Notch1 in SCC and identify the TGFβ-ZEB1-Notch1 axis as potential target for SCC therapy. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To study SCC initiation and progression in vivo, we treated mice with 4-Nitroquinoline 1-oxide (4NQO), a potent oral-esophageal carcinogen.We combined 4NQO treatment with a cell-lineage tracing experimental system in which murine oral and esophageal epithelial basal cells (keratinocytes) were marked permanently with tdTomato fluorescent protein following tamoxifen (TAM)-induced Cre-mediated recombination in K5Cre ERT2 ;R26tdTomato lsl/lsl mice (Fig. 1a).4NQO-induced lesions showed tdTomato accumulation (Fig. 1b; Supplementary Fig. 1a,b), validating the basal keratinocyte origin of these tumors 39 .Flow cytometry revealed the presence of cells displaying both negative and positive expression of EpCAM (EpCAM neg and EpCAM pos ), an epithelial cell surface marker, within the tdTomato-positive (tdTomato pos ) fractions of 4NQO-induced ESCC lesions (Supplementary Fig. 1c), suggesting a loss of epithelial characteristics in tumor cells originating from esophageal basal keratinocytes.In cell lineage tracing experiments, tdTomato expression assures that these EpCAM neg cells are not co-existing intratumoral stromal cells (e.g., fibroblasts) which are not labeled with tdTomato via K5Cre ERT2 .Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis coupled with fluorescence-activated cell sorting (FACS) demonstrated further upregulation of genes associated with mesenchymal cells (Fig. 1c) including Zeb1, in tdTomato pos ;EpCAM neg cells originating from basal esophageal keratinocytes.Consistent with EMT as described in human ESCC 11 , neoplastic lesions displayed E-cadherin loss or mislocalization as well as robust Zeb1 expression at the stromal interface (Fig. 1d).Single-cell-derived three-dimensional (3D) organoids generated from 4NQO-induced dysplastic mucosa and primary tumors recapitulated acquisition of mesenchymal properties as found in 4NQO-induced neoplastic lesions (Fig. 1e).We also included mice with conditional loss or expression of mutant p53 since p53 dysfunction promotes EMT 19,31,40 and found that more pronounced E-cadherin loss and reciprocal Zeb1 upregulation were observed in invasive tumors with mutant p53 R172H (Supplementary Fig. 1d). We next utilized this experimental platform to examine expression of ICN1 (ICN1 Val1744 ), the activated form of Notch1, throughout the natural history of ESCC.In comparison to normal esophageal squamous epithelia, ICN1 expression was increased in dysplastic lesions and remained upregulated in primary and metastatic ESCC (Fig. 1f, g; Supplementary Fig. 1e).Moreover, ICN1 was detected in invasive ESCC cells displaying E-cadherin mislocalization and Zeb1 upregulation (Supplementary Fig. 1d).ICN1 was also expressed highly in neoplastic organoids displaying EMT (Fig. 1e), suggesting a potential role for ICN1 in the acquisition of mesenchymal properties by SCC cells.The tumor suppressor p53 protein can transcriptionally activate Notch1 in response to genotoxic stress 41 .Since 4NQO activates p53 via DNA damage 42 , ICN1 expression in 4NQO-induced early lesions may reflect p53 activation in dysplastic cells.Conversely, Notch1 downregulation may be accounted for by p53 inactivation during disease progression.Therefore, we evaluated the influence of p53 loss upon ICN1 expression in the esophageal epithelium.TAM-induced K5Cre ERT2 -driven p53 loss did not affect ICN1 expression in mice without 4NQO treatment (P = 0.06 by Student's t-test; n = 3 animals per group; two independent experimental replicates), a finding that recapitulates p53 -/- murine epidermis 43 .When p53 was deleted in oral-esophageal keratinocytes then mice were treated with 4NQO, p53 loss did not prevent ICN1 expression in neither normal esophageal epithelium nor 4NQO-induced neoplastic lesions (Fig. 1g; Supplementary Fig. 1e).Mice with p53 deletion did, however, display frequent metastases (Supplementary Fig. 1a).These findings suggest that p53 may be dispensable for Notch1 activation in ) in representative single-cell-derived organoids from normal mucosa or 4NQO-induced tumors.Note that spherical organoids from 4NQO-untreated control mice exhibit a differentiation gradient with predominant luminal keratinization, whereas tumor-derived organoids (neoplastic) display irregular morphology with increased cellularity and diminished keratinization.Zeb1 expression was robust in tumor organoids, particularly at invasive protrusions with decreased E-cadherin expression and detectable ICN1 expression.Box denotes area magnified in panel below.Scale bars, 20 µm.f Representative IHC image for ICN1 4NQO-induced IEN (dysplasia) containing spindle-shaped cells (arrowheads).Scale bar, 50 µm.g Quantification of ICN1 IHC scoring in normal mucosa and 4NQO-induced lesions in mice of indicated genotype.*P < 0.05 vs. p53 +/+ (K5Cre ERT2 ;R26tdTomato lsl/lsl ) normal, # P < 0.0005 vs. p53 -/-(K5Cre ERT2 ;R26tdTomato lsl/lsl ; p53 loxP/loxP ) normal; † P < 0.05 vs. p53 -/-IEN; ns not significant vs. p53 +/+ normal.Data in b-f represent at least three independent 4NQO-induced lesions and >20 organoids from at least two independent experimental replicates.In g, n = 5 normal, n = 8 IEN, and n = 5 invasive ESCC in p53 +/+ esophagi.N.D. not detectable.n = 3 normal, n = 7 IEN, n = 5 invasive ESCC, and n = 4 metastatic tumors in p53 -/-esophagi.Two independent experimental replicates were carried out.Bar diagrams represent mean ± s.d. in c and mean ± s.e.m. in g.Student's t-test was used for paired data comparisons in c, g.ANOVA with Tukey's post hoc test was used for multiple comparisons in g NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01500-9 ARTICLE NATURE COMMUNICATIONS | 8: 1758 | DOI: 10.1038/s41467-017-01500-9 | www.nature.com/naturecommunications4NQO-induced lesions, albeit essential for overall SCC progression. ",
"section_name": "EMT and Notch1 activation are features of carcinogen-driven ESCC in vivo.",
"section_num": null
},
{
"section_content": "To explore further the functional role of Notch1 in ESCC tumorigenicity, we first utilized the extensively characterized human ESCC cell lines TE11 and EN60 12 .Both express ICN1 and form tumors upon xenograft transplantation in immunodeficient mice where doxycycline (DOX)-inducible ectopic ICN1 augments tumor growth 11,12 .To assess Notch activity in vivo, we used 8×CSL-GFP, a lentiviral green fluorescent protein (GFP) reporter driven by the minimal SV40 promoter fused to concatemeric CSL-binding sites (Fig. 2a).In TE11 and EN60 xenograft tumors, a subset of turboRFP (tRFP)-expressing tumor cells exhibited CSL-mediated transcriptional activity (Fig. 2b).A functional role for Notch activity in ESCC tumor growth is evident as dominant negative mutant MAML1 (DNMAML1) 44 prevented DOX-induced ectopic ICN1 from stimulating tumor growth (Fig. 2c).Additionally, DNMAML1 alone or NOTCH1-directed short hairpin RNA (shRNA) suppressed tumor volume significantly (Fig. 2c,d).CRISPR/Cas9-mediated NOTCH1 deletion dramatically impaired tumor formation by TE11 cells in immunodeficient mice (Fig. 2e), supporting a role for Notch1 in tumor initiation as well.2f).Human SCC tumors comprise CD44L and CD44H cells, the latter exhibiting augmented tumor-initiating capability 28,29 .CD44H expression is associated with mesenchymal characteristics in transformed oral-esophageal cell lines 31 .Following FACS-purification, early passage CD44H TE11 and EN60 cells maintained enhanced expression of the mesenchymal cell marker N-cadherin as compared to their CD44L counterparts in culture (Supplementary Fig. 2a).Additionally, CD44H cells isolated from TE11 and EN60 xenograft tumors exhibited upregulation of ZEB1 and CDH2 (N-cadherin) (Fig. 3a; Supplementary Fig. 2b).As ectopic ICN1 expression in EN60 tumors enhanced robustly intratumoral CD44H cell content (Fig. 3b), we hypothesized that Notch1 contributes to tumor initiation via generation of CD44H cells that have mesenchymal properties.To determine how Notch1 may influence tumor initiation by CD44L and CD44H cells, we performed serial transplantation experiments (Fig. 3c).We first grew tumors without activating DOX-inducible ICN1.We then purified CD44L and CD44H cells by FACS from primary tumors and 1 × 10 3 cells were serially injected into recipient mice where DOX was given to induce ectopic ICN1 expression.CD44L cells purified from EN60 and TE11 tumors showed low (<30%) spontaneous tumor formation efficiency; however, ICN1 dramatically stimulated tumor initiation by CD44L cells to 80-90%.In TE11 CD44L cells, DNMAML1 not only antagonized ICN1-mediated tumorigenicity, but also suppressed spontaneous tumor formation.While purified CD44H cells were highly tumorigenic (80-100%), neither ectopic ICN1 nor DNMAML1 affected tumor initiation by CD44H cells, suggesting that established CD44H-mediated tumor initiation is independent of Notch1.To our knowledge, this is the first demonstration that Notch1 may facilitate tumor initiation by converting CD44L cells to highly tumorigenic CD44H cells with mesenchymal traits in vivo. TGFβ and Notch1 cooperate to drive EMT in the tumor microenvironment.The functional role of Notch1 in EMT was suggested as NOTCH1 deletion in cultured TE11 cells attenuated sharply TGFβ-mediated EpCAM neg cell induction (Fig. 3d).Cremediated ex vivo Notch1 deletion in 3D ESCC organoids generated from 4NQO-induced Notch1 loxP/loxP murine tumors resulted in a diminished expression of Zeb1 (Fig. 3e).Moreover, DOXinduced ectopic ICN1 augmented intratumoral EpCAM neg cell content in TE11 xenograft tumors (Supplementary Fig. 2c).While these data support a role for Notch1 in promotion of ESCC tumor cells with attributes of EMT, EpCAM neg cells were rare in cultured TE11 cells and ectopic ICN1 expression alone had no influence upon this cell population (Supplementary Fig. 2c), indicating that Notch1 activation alone may not be sufficient to drive EMT. In the tumor microenvironment, Notch signaling may be modulated by other transcription factors such as HIF1α 17 and attenuated ICN1 expression as well as morphological evidence of EMT in esophageal epithelium (Fig. 4b,c).Taken together, these data indicate that TGFβ functions as a critical positive effector of Notch1 and EMT in the context of the tumor microenvironment. To dissect further the mechanistic role of Notch1 in the generation and maintenance of ESCC tumor cells with mesenchymal features, we utilized the genetically engineered transformed esophageal cell line EPC2T (EPC2-hTERT-EGFR-p53 R175Hcyclin D1), comprising discrete CD44L and CD44H subpopulations with epithelial and mesenchymal traits, respectively 31 (Supplementary Fig. 3a).Under basal conditions, Notch activity, as measured using 8×CSL-GFP reporter (Fig. 2a), was highest within a subset of intermediate transitioning cells (designated as CD44T) as compared to CD44L or CD44H cells (Fig. 5a).In TGFβ-mediated EMT with a resulting increase in CD44T and CD44H cells, Notch activity was augmented further in the CD44T subpopulation (Fig. 5a), indicating that Notch activity may be augmented transiently during the CD44L-to-CD44H transition.Induction of CD44H cells may be accounted for by expansion of pre-existing CD44H cells as well as conversion from CD44L cells.Following FACS purification, CD44H cells failed to display CD44L repopulation even after extended passage (Supplementary Fig. 3b).CD44L cells that were cultured in the absence of TGFβ underwent expansion while permitting minimal CD44H cell repopulation within 5 weeks (four passages) (Supplementary Fig. 3b); however, purified CD44L cells robustly gave rise to CD44H cells upon TGFβ stimulation (Supplementary Fig. 3c; Fig. 5b).A requirement for TGFβ in CD44H cell induction was suggested as pharmacological inhibition of TGFβ receptormediated signaling suppressed spontaneous conversion of purified CD44L cells to CD44H cells (Supplementary Fig. 3d).A permissive role for Notch1 in CD44H cell generation via TGFβ-induced EMT was implicated further as a γ-secretase inhibitor (GSI), DNMAML1 or NOTCH1-directed shRNA each individually attenuated TGFβ-mediated CD44H cell expansion from purified CD44L cells (Supplementary Fig. 3c; Fig. 5b).Ectopic ICN1 augmented CD44H cell expansion from TGFβstimulated purified CD44L cells where ICN1 failed to influence spontaneous CD44H cell expansion in the absence of TGFβ (Fig. 5c).Taken together, these data indicate that Notch1 activation is required, but not sufficient, for EMT-mediated CD44H cell induction in EPC2T cells. Transcriptional repression of Notch3 via Zeb1 permits Notch1mediated EMT.As our findings indicate a role for Notch in EMT, we next sought to determine how Notch1 regulates cell fate in squamous epithelia.The 8×CSL-GFP reporter activity was unaffected in CD44T and CD44H cells upon calcium-mediated squamous-cell differentiation (Fig. 5a) 4 .In CD44L cells, which displayed differentiation in organotypic 3D culture (OTC) when compared to invasive CD44H cells (Fig. 5d), calcium treatment enhanced Notch activity in a subset of cells (Fig. 5a).Thus, local environmental cues may impact Notch signaling to direct cell fate determination.In agreement with this notion, pharmacological inhibition of TGFβ signaling in purified CD44H cells induced CD44L cells with concurrent downregulation of EMT markers and NOTCH3 upregulation (Fig. 5e,f).A trend toward increased Notch3 expression was also detected in peeled murine esophageal epithelia with 4NQO-induced neoplastic lesions following treatment with anti-TGFβ-blocking antibody 1D11 (Supplementary Fig. 3e).Gene array analysis in EPC2T cells revealed further that ectopic ICN1 expression induced a gene expression pattern compatible with squamous-cell differentiation.However, TGFβ treatment in the context of ICN1-overexpression triggered a robust shift toward an EMT-associated gene expression signature (Fig. 5g; Supplementary Fig. 3f) (GSE37994, GSE37993).qRT-PCR analysis confirmed that ICN1 and TGFβ cooperate to induce expression of EMT regulators, including ZEB1 and SNAI1, while TGFβ limited ICN1-mediated expression of NOTCH3 as well as IVL, the latter a marker of terminal differentiation (Fig. 5h).Although the lack of discontinuous CD44L and CD44H cell populations in EN60 and TE11 prevented their isolation for longterm cell culture analyses, the role of Notch and TGFβ signaling in induction of CD44H cells and regulation of squamous-cell differentiation was recapitulated in TE11 cells (Supplementary Fig. 3g-i). We investigated next the mechanistic role of Notch3 in EMT in the context of ESCC.TE11 serial transplantation experiments revealed that DOX-induced ectopic ICN3 suppressed tumor initiation by CD44L cells (Fig. 6a).In EPC2T cells, ectopic ICN3 induced IVL in the presence or absence of TGFβ stimulation (Supplementary Fig. 4a,b).Moreover, ectopic ICN3 abrogated TGFβ-mediated CD44H cell expansion in EPC2T cells (Supplementary Fig. 4c), while NOTCH3 knockdown was sufficient to promote CD44H cell expansion (Fig. 6b) coupled with decreased differentiation and increased EMT characteristics (Fig. 6c) in the absence of TGFβ stimulation.These findings indicate that Notch3, unlike Notch1, may limit EMT so as to permit squamous-cell differentiation.To define the mechanism through which Notch3 expression is suppressed during EMT, we analyzed the NOTCH3 locus by the ECR browser 47 .This analysis predicts two conserved ZEB-binding sites in the NOTCH3 second intron (N3Int2) adjacent to the CSL-binding sites (Fig. 6d) that is occupied by ICN1 during squamous-cell differentiation 4 .Hypothesizing that ZEB transcription factors repress Notch3, we evaluated the influence of ectopic ZEB1 or ZEB2 expression upon N3Int2-modulated transcriptional activity.ZEB1 specifically suppressed basal pGL3-N3Int2-luc reporter activity and also abrogated reporter activation via DOX-induced ectopic ICN1 (Fig. 6e; Supplementary Fig. 4d).Chromatin immunoprecipitation (ChIP) analysis revealed enrichment of ZEB1 binding to the N3Int2 region in purified CD44L cells with TGFβ stimulation and purified CD44H cells without TGFβ stimulation (Fig. 6f).TGFβ did not prevent ICN1 from binding to the N3Int2 region (Supplementary Fig. 4e), suggesting that ZEB1 binding may predominate over ICN1 to repress NOTCH3 transcription.ZEB1-directed shRNA attenuated TGFβ-induced CD44H cell expansion, further implicating ZEB1 as a critical positive regulator in EMT-mediated generation of CD44H cells (Fig. 6g).Notch1 at SCC invasive fronts predicts poor patient prognosis. To demonstrate further the importance of Notch1 activity in pathogenesis of human SCCs, we analyzed surgically procured tissue samples (Supplementary Data 1) by immunohistochemistry (IHC) with two independent NOTCH1 antibodies, both detecting nuclear NOTCH1 at a 95.24% concordance rate (κ = 0.664, 95% CI: 0.008-1.0,Kappa statistic, n = 21, Supplementary Fig. 5).ICN1 (ICN1 Val1744 ) was readily detectable in normal esophageal squamous epithelia (n = 102) as described 4 and was upregulated in superficial precancerous as well as early invasive ESCC lesions (Supplementary Fig. 6).Within tumors invading into submucosa or muscularis propria, most SCC cells (>80%) did not express ICN1 with or without NOTCH1 mutations 6,7 ; however, a small subset of SCC cells expressed ICN1 in the invasive tumor front (35.3% for HNSCC, n = 17; 37.0% for ESCC, n = 227) (Fig. 7a; Supplementary Fig. 6).Accompanied by desmoplastic stroma, ICN1-positive invasive SCC often displayed spindle-shaped cell morphology and ZEB1 co-localization (17.1%, n = 174) (Fig. 7a; Supplementary Fig. 7) with ICN1 and ZEB1 expression being correlated (ρ = 0.31, P < 0.0001, Pearson correlation).When stratified by co-localization status, expression of ICN1 and ZEB1 was relatively uncorrelated for subjects lacking co-localization (ρ = -0.1,P = 0.2, Pearson correlation), while expression was correlated more strongly for those showing co-localization (ρ = 0.37, P < 0.05, Pearson correlation, n = 30).Additionally, these cells were often found invading into lymphatic vessels (Fig. 7b).NOTCH3 expression was low, if not absent, in SCC cells with concurrent ICN1 and ZEB1 expression (Supplementary Fig. 7).Moreover, ICN1 was expressed in a subset of ESCC cells with elevated CD44 expression (Fig. 7c).Evaluation of ICN1 in relation to clinicopathological data revealed that ICN1 was significantly associated with increased lymph node and distant metastases and advanced disease stages (Supplementary Table 1).Finally, analyses of clinical databases revealed that ICN1 expression at the invasive tumor front predicts independently for poor prognosis (Fig. 7d; Supplementary Table 2). ",
"section_name": "Notch1 promotes ESCC tumorigenicity and expansion of CD44H cells with mesenchymal properties.",
"section_num": null
},
{
"section_content": "The current study highlights an oncogenic role for Notch1 in SCC via EMT-mediated induction of CD44H cells with enhanced malignant potential (Fig. 7e).We demonstrate that Notch1 and Notch3 may have opposing functions to allow expansion of CD44H cells both in vitro and in vivo.Moreover, we find that TGFβ guides Notch1 to drive EMT via a previously undescribed robust shift in the spectrum of Notch1 target genes including Notch3, which is essential in squamous-cell differentiation 4 and senescence 12,48 .Given the microRNA-mediated crosstalk between Notch signaling and ZEB1 33,49 , our findings indicate that direct repression of NOTCH3 by ZEB1 is a novel mechanism through which ZEB1 may influence Notch1-mediated cell fate determination.Activated Notch1 (i.e., ICN1) interacts with TGFβ downstream effector SMAD3 45 .ZEB1 also binds physically to SMAD3 to enhance TGFβ-mediated transcription 34 .Additionally, ZEB1 promotes tumorigenicity by repressing stemnessinhibiting microRNAs in pancreas cancer 50 , where ZEB1 induction and EMT have been implicated as the earliest event preceding tumor formation in cell-lineage tracing experiments 51 .Taken together, these preclinical studies suggest that Notch1 and ZEB1 may cooperate to promote carcinogenesis and SCC progression via TGFβ-mediated EMT and offer the TGFβ-ZEB1-Notch1 axis as potential therapeutic target in SCC.Despite a well-established role for the Notch pathway in regulation of varied cell fates in a tissue-dependent and contextdependent manner, the molecular mechanisms governing Notchmediated cell fate determination have yet to be fully elucidated.In non-transformed esophageal epithelium, Notch1-dependent transcriptional upregulation of Notch3 mediates squamous-cell differentiation as shown by us 4 .In the current study, we find Notch1 facilitates EMT-mediated expansion of CD44H cells with enhanced malignant potential.This raises the question of how Notch1 may act as a signal to induce both differentiation and dedifferentiation (i.e., EMT) in squamous epithelium.One potential explanation for this dichotomy is the influence of tissue microenvironment upon Notch-mediated cell fate determination.Indeed, microarray gene expression analysis in ICN1overexpressing transformed esophageal keratinocytes indicates that TGFβ induces a dramatic shift in the spectrum of Notch1 target genes from a profile enriched for genes involved in squamous-cell differentiation to one consistent with EMT.Notch1 may also facilitate senescence in response to TGFβ in esophageal keratinocytes with intact cell cycle checkpoint functions 12 .The complexity in the role of Notch signaling in tumor biology is echoed by that of TGFβ as intratumoral cancer cell heterogeneity represents cancer cells that can respond to TGFβ and those cannot, the latter emerging during disease progression 52 .Thus, like TGFβ, Notch1 may have differential roles in cancer initiation and development.Besides TGFβ, hypoxia and inflammation may activate Notch1 to promote EMT in the tumor microenvironment via transcriptional factors such as HIF1α 17 and NF-κB 53 , respectively.Potential influence of these factors upon Notch1-mediated cell fates warrants further investigation in SCC as well as squamous epithelia under eosinophilic esophagitis where EMT and Notch3 downregulation are implicated (P.M.C. and H.N., unpublished observation). While our own published findings and those of others have implicated Notch1 as a tumor promoter in SCCs 12,[54][55][56] , the current study is the first to demonstrate in vivo that Notch1 facilitates tumor initiation by converting CD44L cells to highly tumorigenic CD44H cells with mesenchymal traits, complementing the earlier studies implicating EMT and ZEB1 in the CD44L-to-CD44H transition 24,28,57 .While Notch1 activation and EMT were detected at the invasive tumor front, SCC cells with epithelial characteristics were commonly present in xenograft tumors, 4NQO-induced tumors, and human SCC samples.In our cell-lineage tracing experiments utilizing K5Cre ERT2 ; R26tdTomato lsl/lsl mice carrying 4NQO-induced tumors, complete loss of epithelial characteristics was uncommon, albeit present (Supplementary Fig. 1d).Despite enhanced expression of mesenchymal-associated genes in EpCAM neg cells (Fig. 1c,d), most tumors displayed a broad range of continuous EpCAM expression (Supplementary Fig. 1c), suggesting a highly heterogeneous and plastic nature of SCC cells.Besides squamous-cell differentiation, Notch3 may be permissive for mesenchymal-epithelial transition or epithelial-mesenchymal hybrid characteristics of cancer cells by limiting EMT 58,59 .These findings have potential clinical impact as reducing CD44H cells may delay disease progression or post-therapeutic recurrence and increase sensitivity to chemotherapeutic agents, providing a platform for new human clinical trials.Given the interplay between Notch1 and Notch3 in regulating esophageal cell fate, the use of pan-Notch inhibitors may prove problematic.Indeed, genetic Notch inhibition via DNMAML1 expression accelerates carcinogen-mediated esophageal tumor initiation, growth and invasion in vivo 60,61 .Tumor initiation by CD44H cells may be independent of Notch because purified CD44H cells gave rise to tumors with or without DOX-induced ICN1 expression or in the presence of DNMAML1 (Fig. 3c).Interestingly, Notch inhibition by DNMAML1 permits clonal immortalization and expansion of murine esophageal epithelial cells 61 .Thus, Notch1 activation is essential for the induction of CD44H cells while it may be dispensable for their maintenance and tumor initiation by CD44H cells.Such a premise is corroborated by ICN1 expression localized to tumor invasive fronts, but not in the majority of expanding ESCC cell populations (Supplementary Fig. 6).Notch signaling can be selectively modulated by Notch receptor paralog-specific antibodies 62,63 .TGFβ signaling can be also regulated by TGFβ inhibitors 64 .Notably, the observed inhibitory effects of anti-TGFβ antibody upon tumor progression in mice (Fig. 4) may involve TGFβ downstream effectors other than Notch1, representing a limitation of the current study.Since TGFβ-induced EMT involves autophagy in ESCC cells where pharmacological inhibition of autophagy flux by chloroquine decreases CD44H cells, an alternative approach to target EMT and CD44H cells 30 .Future studies evaluating the efficacy of these agents in SCC therapy are currently underway in preclinical models. Technical innovation in this study includes use of a HNSCC PDX model as well as a cell-lineage tractable mouse model of ESCC through which EMT was for the first time documented unequivocally in SCC cells in situ.Additionally, we have developed an esophageal 3D organoid platform that recapitulates esophageal tissue architecture ex vivo and can be utilized to model esophagitis 65 .Here, we apply this model system to SCC, generating murine ESCC-derived 3D organoids for the first time (Fig. 1e).Upon ex vivo culture, single-cell-derived 3D organoids from 4NQO-induced dysplasia and ESCC tumors display morphological characteristics of neoplastic epithelium, including nuclear atypia, perturbed squamous-cell differentiation and evidence of EMT, which are maintained upon organoid passaging (K.A.W., P.M.C., and H.N., unpublished observation).We have also successfully performed ex vivo Cre-mediated recombination in 3D organoids generated from neoplastic esophageal keratinocytes of 4NQO-treated Notch1 loxP/loxP mice, demonstrating that Notch1 is required for organoid formation and EMT-like features in neoplastic 3D organoids (Figs.2f,3e).Notch1 deletion in the murine skin promotes SCC development in a non-cell autonomous fashion due to inflammation associated with epidermal barrier defects 5 , limiting the assessment of the cell-autonomous oncogenic role of Notch1 in vivo.The 3D organoid system provides insights about cell-autonomous oncogenic functions of Notch1 in the absence of inflammatory milieu ex vivo, complementing in vivo experiments.The 4NQO model requires a long-term 4NQO administration (16 weeks) where tumors arise 6-9 weeks after 4NQO withdrawal during the observation period; however, it is not precisely known when malignant transformation occurs in this model.The single-cell-derived 3D organoids have a potential to detect neoplastic changes in a more sensitive and quantitative manner than conventional morphological tissue assessment.Such a study is underway.Once an appropriate window of 4NQO-induced malignant transformation is determined, our cell-lineage traceable mice can be utilized for genetic ablation of Notch1 following 4NQO administration to elucidate how Notch1 may exert its oncogenic role by promoting EMT and tumor initiation and/or progression in vivo.Alternatively, such mice may be treated with Notch1-specific antagonistic antibody 63 or anti-Notch3 agonistic antibody 62 , the latter can be used for targeted activation of Notch3.While these findings identify 3D neoplastic organoids as sustainable resource for functional and mechanistic investigations into the biology of ESCC, it remains to be determined how faithfully organoid genetics and biology mimic that of the tissue from which they are derived upon extended ex vivo culture.Ongoing studies include procuring of a bank of human patient-derived ESCC 3D organoids that may be utilized as an experimental platform for discovery and validation of novel translational applications for prognosis and therapy in the setting of personalized medicine. Taken together, the current study supports a tumor-promoting role of Notch1 in SCC while also providing novel mechanistic insight into how the local tissue microenvironment may influence Notch-mediated cell fate determination. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Patients and tissue samples.Surgically removed tissue samples (Supplementary Data 1) were described previously 11,[66][67][68] or newly procured at Kagoshima University Hospital in accordance with Institutional Review Board standards and guidelines.Informed consent was obtained from all human subjects.Most samples were available as tissue microarrays (TMAs) 67,68 containing primary ESCC (n = 171), carcinoma in situ (CIS; n = 9), IEN (n = 7), and normal mucosa (n = 114).Samples with NOTCH1 mutations (n = 4; E755*, D469Y, R1279D, and D1457G) and wild-type NOTCH1 (n = 15) were identified by DNA sequencing (SRP072948) 69 .Given the limitation of TMAs to assess intratumoral cancer cell heterogeneity 70 , we examined whole paraffin blocks (n = 244) by Hematoxylin and Eosin (H&E) staining and IHC, as described below, following preliminary analysis with TMAs.Survival analysis was done on IHC data for 185 ESCC patients who did not receive chemotherapy or radiation therapy prior to surgery. Esophageal epithelial cell-lineage traceable mice and 4NQO treatment.The K5Cre ERT2 transgenic mouse strain 68 was intercrossed with R26tdTomato lsl/lsl (Jackson Laboratory, Bar Harbor, ME) carrying the homozygous Rosa26 locus with knocked-in tdTomato fluorescent protein as a reporter under the loxP-stop-loxP sequence.The resulting K5Cre ERT2 ;R26tdTomato lsl/wt mice were crossed with R26tdTomato lsl/lsl mice to generate K5Cre ERT2 ;R26tdTomato lsl/lsl mice.Similarly, K5Cre ERT2 ;p53 loxP/loxP mice were generated with p53 loxP/loxP mice (Jackson Laboratory).R26tdTomato lsl/lsl mice were also crossed with p53 loxP/loxP to generate R26tdTomato lsl/wt ;p53 loxP/wt mice, which were then crossed to generate R26tdTomato lsl/lsl ;p53 loxP/loxP mice.The K5Cre ERT2 ;p53 loxP/loxP mice were further crossed with R26tdTomato lsl/lsl ;p53 loxP/loxP mice to generate K5Cre ERT2 ;R26tdTomato lsl/wt ; p53 loxP/loxP .These mice were further crossed with R26tdTomato lsl/lsl ;p53 loxP/loxP to generate and maintain the K5Cre ERT2 ;R26tdTomato lsl/lsl ;p53 loxP/loxP strain.The L2Cre transgenic mouse strain 71 was intercrossed with p53 R172H/R172H mice 72 to generate L2Cre;p53 R172H/wt mice. We administered TAM (Sigma-Aldrich, St. Louis, MO; 0.25 mg/g body weight) via oral gavage to 3-4-month-old K5Cre ERT2 ;R26tdTomato lsl/lsl and K5Cre ERT2 ; R26tdTomato lsl/lsl ; p53 loxP/loxP littermates 2 weeks before starting 4-Nitroquinoline N-oxide (4NQO) (Sigma-Aldrich) treatment.Mice received 100 µg/ml 4NQO in 2% propylene glycol (MP Biomedicals, Solon, OH) in drinking water for 16 weeks and were followed up for 8-10 weeks (or earlier if mice were ill) after 4NQO withdrawal as described 73 .To inhibit TGFβ receptor-mediated signaling, mice were treated for 2 weeks with either anti-TGFβ antibody 1D11 (intra-peritoneally), neutralizing all three isoforms of TGFβ 46 (a gift of Dr. Singhal, University of Pennsylvania; 3 mg/kg, three times per week) or control IgG (intra-peritoneally) with injections initiated 4 weeks following 4NQO withdrawal.At the time of sacrifice, one half of the dissected esophagus or a tumor (if visible macroscopically) from each mouse was fixed in 4% paraformaldehyde and paraffin-embedded for morphological analyses.The other half of esophagi and tumors was dissociated as described previously 74 and cell suspensions were subjected to flow cytometry or FACS and organoid formation assays.All experiments were done under approved protocols from the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC).Sample size for groups were projected based on data from pilot studies.Animals were only excluded from analyses in event of death from procedure-related causes (e.g., death following oral gavage) that were unrelated to experimental differences between groups.Animals were randomized to treatment groups with consideration given to representation of both sexes.Investigators were informed of groups during treatment phase of experiments.Upon processing, tissues were given a unique identifier to blind investigators during analyses and outcome assessments. Xenograft transplantation experiments.Xenograft transplantation experiments were done as described 11,12 .In brief, 1-5 × 10 6 cells were suspended in 50% Matrigel and implanted subcutaneously into the dorsal skin of female athymic nu/ nu mice (4-6 weeks old; Charles River Breeding Laboratories).PDX tumor transplantation was performed as previously described with fragments of passaged PDX tumors implanted under the dorsal skin of NOD/SCID/IL2 receptor γ-chaindeficient (NSG) mice 75 .Tumor growth was monitored and mice were sacrificed 6-10 weeks after inoculation for flow cytometry and histopathological analyses.One half of each tumor was fixed in formalin and embedded in paraffin blocks.The other half of each tumor was enzymatically dissociated and subjected to flow cytometry and FACS for serial transplantation experiments where 10-10 3 cells were injected into recipient mice to determine tumor formation rates.For tetracycline/DOX-inducible (TetOn) transgene expression in xenograft tumors, mice were fed daily with DOX-containing pellets (20 mg/kg).To inhibit TGFβ receptormediated signaling, mice were treated for 2 weeks with either anti-TGFβ antibody 1D11 (intra-peritoneally) (3 mg/kg, 2-3 times per week) or control IgG (intraperitoneally), beginning when tumor volume reached 60 mm 3 .All experiments were approved by the University of Pennsylvania IACUC or under Wistar Institute IACUC protocols 112652/112655.Sample size for groups were projected based on previous xenograft studies then adjusted following acquisition of data in initial ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01500-9 experiment.Animals were only excluded from analyses in event of death from procedure-related causes (e.g., sepsis) that were unrelated to experimental differences between groups.Animals were randomized by cage upon arrival.Investigators were informed of groups during treatment phase of experiments.Upon processing, tumors were given a unique identifier to blind investigators during analyses and outcome assessments.DNA constructs.A lentiviral vector pTRIPZ expressing DOX-inducible shRNA directed against human NOTCH1 (Notch1-A; V2LHS_149557 and Notch1-B; V3LHS_637133) and pTRIPZ-NS carrying a non-silencing scrambled sequence (RHS4346) (GE Dharmacon, Lafayette, CO) were purchased.Other shRNA sequences in pGIPZ (GE Dharmacon) targeting NOTCH3 (Notch3-A; V2LHS_229748 and Notch3-B; V2LHS_93017), ZEB1 (ZEB1-A; V2LHS_116663 and ZEB1-B; V2LHS_116659), and a non-silencing scrambled sequence (RHS4348) were used as described previously 11,12,19 . To generate a lentiviral vector pTR-tRFP expressing constitutively turbo RFP (tRFP) under ubiquitin C (UBC) promoter, a DOX-inducible gene expression vector pTRIPZ-MCS 12 was modified by removal of BamHI and LoxP sites and a subsequent insertion of new multiple cloning sites (MCS) comprising 5′-AgeI-BamHI-SnaBI-EcoRI-XhoI-MluI-3′, resulting in the creation of pTRIPZ-MCS2.We then amplified an open reading frame (ORF) for turbo RFP (tRFP) by PCR using pTRIPZ-NS (RHS4743, GE Dharmacon) as a template with primers 5′-AGCGCTAGCGCCACCATGAGCGAGCTGATC-3′ and 5′-AGCGCGGCCGC TTATCTGTGCCCCAGTTTGCTAGG-3′.Following removal of rtTA-encoding ORF flanked by NheI and NotI sites, the purified tRFP ORF was subcloned into the NheI and NotI sites of pTRIPZ-MCS2, resulting in creation of pTR-tRFP.To generate a lentiviral vector carrying 8×CSL-GFP reporter, an ORF for GFP was isolated from pBABE-zeo-GFP 19 and ligated into the EcoRI and XhoI sites of pTR-tRFP, generating pTR-tRFP-GFP.A DNA fragment containing a concatemer of eight copies of CSL-binding sites fused to minimal SV40 (mSV40) promoter was isolated from 8×CSL-luc 76 by PCR with primers 5′-AGCTCTAGACTCTTACGC GT GCTAGCTCGA-3′ and 5′-AGCACCGGTTTTGCAAAAGCCTAG GCCTCC A-3′ to replace the sequence containing tetracycline response elements and minimal CMV promoter from pTR-RFP-GFP at the XbaI and AgeI sites, resulting in a creation of pTR-RFP-8×CSL-GFP. To delete human NOTCH1 via Crispr/Cas9-mediated gene editing, five candidate guide RNA (gRNA) sequences were subcloned into lentiCRISPR v1 (gift of a gift from Feng Zhang; Addgene, plasmid #52961) at its BsmBI sites and used as described 77 .gRNAs 1, 2, and 5 targeting chromosome 9 NOTCH1 exon 14 (EGFlike region at 139,407,894-139,407,913) or exon 19 (EGF-like region at 139,403,465-139,403,484) and exon 34 (PEST domain at 139,390,539-139,390,558), respectively, were found to be equally effective to delete NOTCH1. To generate a retroviral vector BABE-zeo-FLAG-DNMAML1, we amplified an Flag-MAML1 ORF by PCR using pFLAG-CMV2-MAML1 (1-302) (a gift from Dr. Wu, University of Florida) as a template with primers 5′-GCACGGAT CCGCCACCATGGACTACAAAGACGATGA CGACAAG-3′ and 5′-GCACGTC GACCTAGAATTCCGTCTTAATATTAATGTCCTGTGCC-3′ and subcloned at the BamHI and SalI sites of pBABE-zeo 78 .To generate pBABE-bla-ZEB1 and pBABE-bla-ZEB2, ZEB1 and ZEB2 ORFs in pCR-BluntII-TOPO were purchased from GE Dharmacon (clone 40036600 for ZEB1 and clone 40124124 for ZEB2).The ZEB1 and ZEB2 ORFs were amplified by PCR with primers 5′-CGCTGGA TCCGCCGCCACCATGGCGGATGG CCCCAGGTG-3′ and 5′-CGGAGTCGAC TTAGGCTTCATTTGTCTTTTC-3′ for ZEB1 and 5′-CG CTGGATCCGCCGC CACCATGAAGCAGCCGATCATGGC-3′ and 5′-CGGAGTCGACTTACATGC CATCTTCCATATTG-3′ for ZEB2, respectively, and subcloned at the BamHI and SalI sites of pBABE-bla 79 . The 2nd intron of NOTCH3 (N3Int2) containing ZEB1-binding sites (488-bp) was amplified by PCR using the input DNA sample from ChIP assay as a template with primers 5′-GGGAGATCTGGTTCCCACGCTCTGCGTCC-3′ and 5′-GGGC TCGAGCCGCGCCTGGAATACTGCCG-3′.Then the amplified DNA was subcloned at the XhoI and BglII sites of pGL3 promoter (Promega, Madison, WI), creating a plasmid pGL3-N3Int2-luc.All constructs were verified by DNA sequencing. Cell culture, genetic modifications, and pharmacological treatments of cells.TE11, EN60, EPC2T, and derivatives were grown at 37 °C in a 5% CO 2 atmosphere as described previously 12 .Additional genetic modifications were done via stable gene transfer of the above retroviral and lentiviral constructs as described previously 12 .Stable cell lines were established by drug selection for 7 days with 1 µg/ml of Puromycin (Invitrogen) for genes transduced via pTRIPZ or lentiCRISPR; and 0.5 µg/ml zeocin (Invitrogen) for Flag-MAML1 transduced via pBABE-zeo. Cels transduced with GFP (pGIPZ) or tRFP (pTR-tRFP) were selected for the brightest level of fluorescence (top 20%) by FACS.EPC2T, EN60, and derivatives were kept undifferentiated in Keratinocyte serum-free medium (Invitrogen, Carlsbad, CA) containing a low concentration (0.09 mM) of CaCl 2 .Squamous-cell differentiation was induced by 0.6 mM CaCl 2 (Sigma-Aldrich) as described 4 .Compound E (Calbiochem, La Jolla, CA), a GSI, and SB431542 (Calbiochem), a TGFβ type I receptor kinase inhibitor were reconstituted in dimethyl sulfoxide (DMSO) (Sigma-Aldrich).Cells were treated with 1 µM Compound E or 5 ng/ml recombinant human TGFβ1 (R&D Systems, Minneapolis, MN) as optimized previously 4,19,80 .SB431542 was used at 10 µM.Dox was used at 1 µg/ml for DOXinducible transgene expression 12 .Phase contrast images were acquired using a Nikon Eclipse E600 microscope.EPC2-hTERT cells and derivatives were established and extensively characterized by us 12,30,74 .TE11 12,30,81 and EN60 12,82 cells were generous gifts from Dr. Tetsuro Nishihira (Tohuko University, Sendai, Japan) and Dr Hiroshi Shirasawa (Chiba University, Chiba, Japan), respectively, and were extensively characterized by us 12,30,74,83 .The earliest frozen stocks of all cell lines have been stored at the Cell Culture Core of the University of Pennsylvania.We have propagated cells from frozen stocks of original vials that were authenticated by short tandem repeat profiling (ATCC) for highly polymorphic microsatellites to validate the identity of cells by comparing cells at the earliest stocks and those grown >8-12 passages.All cell lines undergo routine mycoplasma testing. Organotypic 3D culture.OTC was carried out as described previously 74 .In brief, 0.5 × 10 6 of epithelial cells were seeded on top of the collagen/Matrigel matrices containing FEF3 human fetal esophageal fibroblasts, and grown in submerged conditions for 4 days.Cultures were then raised to the air-liquid interface for additional 4 days and harvested for morphological assessment.Each OTC experiment was performed in triplicate. Ex vivo esophageal organoid 3D culture.Esophageal keratinocytes were isolated from vehicle-treated or 4NQO-treated K5Cre ERT2 ;R26tdTomato lsl/lsl or Notch1 loxP/loxP (Jacskon Laboratories) mice under an IACUC-approved protocol as described previously 65,84,85 .Using 24-well plates, 5000 cells were seeded per well in 50 μl Matrigel.After solidification, 500 μl of DMEM/F12 supplemented with 1× Glutamax, 1× HEPES, 1× N2 Supplement, 1× B27 Supplement, 0.1 mM N-acetyl-Lcysteine (Sigma-Aldrich), 50 ng/ml mouse recombinant EGF (R&D Systems), 2.0% Noggin/R-Spondin-conditioned media and 10 μM Y27632 (Tocris Biosciences, Bristol, UK) were added and replenished every other day.For ex vivo recombination, organoids were cultured in the presence of Adenovirus vector containing Cre recombinase and GFP (University of Iowa Gene Transfer Vector Core).Adenovirus vector containing GFP alone was used as a control.Adenovirus vectors were used at 1:500 at the time of organoid plating.Organoid formation rate was calculated as the percentage of the number of organoids formed at day 7 per total number of cells seeded at day 0. After 14 days organoids were recovered by digesting Matrigel with Dispase I (BD Biosciences, San Jose, CA; 1 U/ml) and fixed overnight in 4.0% paraformaldehyde.Specimens were embedded in 2.0% Bacto-Agar: 2.5% gelatin prior to paraffin embedding. RNA isolation, cDNA synthesis, qRT-PCR, and microarray analyses.RNA isolation, cDNA synthesis, and qRT-PCR were done using StepOnePlus™ Real-Time PCR System (Applied Biosystems) by TaqMan ® Gene Expression Assays (Applied Biosystems) for NOTCH1 (Hs01062014_m1), Notch1 (Mm00435249_m1), NOTCH3 (Hs00166432_m1), Notch3 (Mm00435270_m1), IVL (Hs00846307_s1), CK13 (s00999762_m1), CDH1 (Hs00170423_m1), CDH2 (Hs00983062 _m1), ZEB1 (Hs00232783_m1), SNAI1 (Hs00195591_m1), and PAI1 (Hs01126606_m1), and SYBR ® Green PCR for human ACTB (β-Actin) as well as murine Cdh1 (5′-TCAAGCTCGCGGATAACCAGAACA-3′ and 5′-ATTCCCGCCTTCATGCAGTTGTTG-3′), Cdh2 (5′-ATGGCCTTTCAAACACA GCCACAG-3′ and 5′-ACAATGACGTCCACCCTGTTCTCA-3′), Zeb1 (5′-TGAG CACACAGGTAAGAGGCC-3′ and 5′-GGCTTTTCCCCAGAGTGCA-3′), Zeb2 (5′-TGATAGCCTTGCAAACCCTCTGGA-3′ and 5′-TCCTTCATTTCTTCTGG ACCGGCT-3′), Twist (5′-AGCTGAGCAAGATTCAGACCCTCA-3′ and 5′-TGC AGCTTGCCATCTTGGAGT-3′), and Gapdh (5′-GGTGGTCTCCTCTGACTTC AACA-3′ and 5′-GTTGCTGTAGCCAAATTCGTTGT-3′) and as described 4,11,19 .All PCR reactions were performed in triplicate.The relative level of each mRNA was normalized to ACTB (β-actin) for human genes and Gapdh for murine genes as internal controls.Gene array experiments were done using an Affymetrix gene chip (U133+v2.0)(Affymetrix, Santa Clara, CA) as described 11,31 .Data were deposited at the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) (accession #GSE37994). Immunoblot analysis.Whole cell lysates were prepared as described 4,11 .20 μg of denatured protein was fractionated on a NuPAGE Bis-Tris 4-12% gel (Invitrogen).Following electrotransfer, Immobilon-P membranes (Millipore) were incubated with primary antibodies for rat monoclonal anti-NOTCH1 5B5 (1:1000; 3447, Cell Signaling, Danvers, MA), rabbit monoclonal anti-ICN1 Val1744 D3B8 (1:1000; 4147, Cell Signaling), rat monoclonal anti-NOTCH3 8G5 (1:1000; 3446, Cell Signaling), mouse monoclonal anti-E-cadherin (1:10,000; 610182, BD Biosciences), mouse monoclonal anti-N-cadherin clone 32 (1:1000; 610920, BD Biosciences), mouse monoclonal anti-Involucrin clone SY5 (1:1000, I9018, Sigma-Aldrich) and mouse monoclonal anti-β-actin (1:10,000; AC-74, Sigma-Aldrich), and then with the appropriate HRP-conjugated secondary antibody (GE Healthcare, Piscataway, NJ).β-actin served as a loading control.Uncropped images of blots from primary figures are shown in Supplementary Fig. 8. Flow cytometry and FACS.Flow cytometry and FACS were performed as described previously 86 .FACSCalibur or LSRII (BD Biosciences, San Jose, CA) and FlowJo (Tree Star, Ashland, OR) were used for flow cytometry.FACS Vantage SE and FACS Aria II (BD Biosciences) were used to sort purified CD44 high -CD24 low/- cells (CD44H) and CD44 low/--CD24 high cells (CD44L) from EPC2T, TE11 and EN60 cells, and/or xenograft tumors.Cells were suspended in Hank's balanced salt solution (Invitrogen) containing 1% BSA (Sigma-Aldrich) and stained with the following antibodies on ice for 30 min: human PE/Cy7-anti-CD24 clone [ML5] (1:40; 311,120, BioLegend, San Diego, CA), human APC-anti-CD44 clone G44-26 (1:20; 559,942, BD Biosciences), human FITC-anti-CD326 (EpCAM) clone HEA-125 (1:50; 130-089-113, Miltenyi Biotec, San Diego, CA) and/or mouse APC-Cy7anti-CD326 (EpCAM) Clone G8.8 (1:50; 118,217, BioLegend).4′,6-diamidino-2phenylindole (DAPI; 2 μg/μl; Invitrogen) was used to assess viability.CD44T cells were defined as CD44 high -CD24 High .To purify CD44L and CD44H cells from TE11 and EN60 xenograft tumors, tumors were minced into 1 mm 3 pieces and incubated in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen) containing 1 mg/ml collagenase I (Sigma-Aldrich) at 37 °C for 90 min.Following centrifugation, residual tissue pieces were digested in 0.05% trypsin-EDTA (Invitrogen) at 37 °C for 10 min and then with 1 U/ml Dispase (BD Biosciences) and 100 μg/ml DNase I (#10104159001, Roche) at 37 °C for 10 min.Dissociated tumor cells were filtered with a 40 µm cell strainer (BD Biosciences) and washed prior to incubation with antibodies.Cancer cells were distinguished from host-derived stromal cells by tRFP expression detected concurrently.In 8×CSL-GFP reporter assays, tRFPpositivity was used to identify human cells while GFP expression was monitored to evaluate Notch activity.In tissue from 4NQO-treated mice and controls, tdTomato expression was used to isolate epithelial cells that had undergone K5Cre ERT2mediated recombination following TAM treatment.Following dissection, esophagi were incubated with 1 U/ml Dispase for 5 min at 37 °C then epithelial layer was peeled from underlying stroma using fine tipped forceps.Epithelia were then incubated in 0.25% trypsin two times for 5 min at 37 °C with agitation to liberate keratinocytes.Trypsin was quenched with soybean trypsin inhibitor then liberated cells were filtered with a 40 µm cell strainer prior to staining.Flow cytometry was repeated for each genotype and condition at least three times. Immunofluorescence (IF) and IHC.IF and IHC for NOTCH1, NOTCH3, ZEB1, E-cadherin, CD44, and tdTomato were done as described previously 4,11,12 .IHC for Notch1 was done with two independent anti-NOTCH1 antibodies, namely, polyclonal rabbit polyclonal anti-NOTCH1 (ab27526; Abcam; 1:250) and rabbit polyclonal anti-ICN1 V1744 (2421; Cell Signaling; 1:100), both detecting cytoplasmic and nuclear expression of NOTCH1.The following primary antibodies were used for IHC and/or IF: rabbit polyclonal anti-NOTCH3 (ab23426; Abcam; 1:250), rabbit polyclonal anti-ZEB1 (a generous gift from Dr. Darling, University of Louisville; 1:100), mouse monoclonal anti-E-cadherin (610182; BD Biosciences; 1:100), rabbit polyclonal anti-RFP (ab34771, Abcam; 1:100).IHC signals were developed using the diaminobenzidine substrate kit (Vector Laboratories, Burlingame, CA) following incubation with secondary anti-mouse IgG (Vector; 1:600 at 37 °C for 30 min) or anti-rabbit IgG (Vector; 1:600 at 37 °C for 30 min), and counterstained with Hematoxylin (Fisher Scientific CS401-1D).For IF, Cyanine Cy2-conjugated or Cyanine Cy3-conjugated affinity-purified anti-mouse or antirabbit IgG (Jackson Immuno-Research; 1:600) was used for signal detection by incubating at 37 °C for 30 min, and cell nuclei were counterstained by DAPI (Invitrogen; 1:10,000).Stained objects were examined with a Nikon E600 microscope and imaged with a digital camera.The staining was assessed independently by pathologists (S.N. and A.K.S.), and the intensity was expressed as negative (0), marginally positive (0.5), weakly positive (1), moderately positive (2), or strongly positive (3).Both antibodies detect cytoplasmic and nuclear NOTCH1 at high concordance rates, although anti-ICN1 V1744 antibody was more sensitive than ab27526 to detect nuclear Notch1 expression (Supplementary Fig. 5).The ab27526 antibody recognizes a full-length NOTCH1 expressed on the cell surface in normal squamous epithelia 4 as well as well-differentiated SCC cells forming keratin pearl 11 .In this study, we have focused on cytoplasmic and nuclear intracellular Notch1 (ICN1) detected by anti-ICN1 V1744 . Transient transfection and dual-luciferase assays.Transient transfection of reporter plasmids and luciferase assays was performed as described previously 4,80 . 1 × 10 5 cells were seeded per well in 24-well plates 24 h before transfection.Lipofectamine TM LTX and Plus TM reagents (Invitrogen) were used for DNA transfection, according to the manufacturer's instructions.400 ng of pGL3p-N3Int2 was transfected along with or without 400 ng of pBABE-bla-ZEB1, pBABEbla-ZEB2 of pBABE-bla (empty vector control).5 ng of phRL-SV40-renilla luciferase vector (Promega) was co-transfected to calibrate the variation of transfection efficiencies among wells.Cells were incubated in the presence or absence of 1 µg/ml DOX to induce ICN1 in cells expressing ICN1 TetOn for 48 h before cell lysis.Alternatively, 5 ng/ml TGFβ1 was added at 24 h after transfection and incubated for an additional 48 h before cell lysis.Luciferase activities were determined using Dual-Luciferase TM Reporter Assay system (Promega) and ORION Microplate Luminometer (Berthold Detection Systems, USA, Oak Ridge, TN).The mean of firefly luciferase activity was normalized with the co-transfected renilla luciferase activity.Transfection was carried out at least three times, and variation between experiments was not greater than 15%. ChIP assay. 1 × 10 7 cells were treated with 1% formaldehyde for 10 min at 37 °C and quenched with 0.125 M glycine for 5 min at room temperature.Cross-linked chromatin was sheared into ~500 bp DNA fragments with Branson Sonifier 250 (Branson, Danbury, CT, USA) and subjected to immunoprecipitation with 2 µg/10 6 cells with antibody for ZEB1 (sc10572, Santa Cruz), NOTCH1 (sc6014-R, Santa Cruz) or either goat IgG (sc2028, Santa Cruz) or rabbit IgG (sc2027, Santa Cruz) as negative controls.DNA was purified by QIAquick PCR purification kit (QIAGEN, Valencia, CA) and analyzed by real-time PCR using StepOnePlus™ Real-Time PCR System (Applied Biosystems, Carlsbad, CA).The following primers were used for real-time qPCR: 5′-CCCACAGCCCAACTCGGAGG-3′ and 5′-CCGCGCCTGG AATACTGCCG-3′ for ZEB1-binding sites at the 2nd intron of NOTCH3, 5′-GC TGGGCGCCGAGGATAG-3′ and 5′-AGACCTCGTCCCCATCTCCTAGTC-3′ for CSL-binding sites 4 at the 2nd intron of NOTCH3, 5′-TGCCAAAAGAGGAAG CATAAGTA-3′ and 5′-TCAAAATCCCTGTGTAGCTGAAT-3′ for an off-target control 4 for NOTCHotch3.5′-CCCTTTCTGATCCCAGGTCT-3′ and 5′-GACCT GCACGGTTCTGATTC-3′ for a ZEB1-binding site on the of CDH1 promoter 87 , 5′-GAAGTGGCTCCAGTGCTCAAA-3′ and 5′-ATGGCAGTGCATGCCTGTA GT-3′ for an off target control for CDH1 promoter 87 , 5′-CGTGTCTCCTCCTC CCATT-3′ and 5′-CCGCTGTTATCAGCACCAG-3′ for a CSL-binding site at the HES1 promoter 88 , 5′-TGGATCCAATCCTATTGCCC-3′ and 5′-CGCAGCAGT TGGAAGTGTTT-3′ for an off target control for HES1 88 .Data represent three independent experiments. ",
"section_name": "Methods",
"section_num": null
}
] |
[
{
"section_content": "We thank Ben Rhoades, Medha Sharma, and the staff of the Molecular Pathology and Imaging Core, Molecular Biology/Gene Expression Core, Cell Culture Core and Transgenic and Chimeric Mouse Core Facilities for technical support.We thank members of the Nakagawa lab, the Rustgi lab, and the Basu lab for helpful discussions.P.M.C. is currently in the Graduate program in Cell Biology, Physiology, and Metabolism at the University of Pennsylvania.This study was supported by the following National Institute of Health (NIH) Grants: National Cancer Institute, P01CA098101 (to H.N., M.N., K.A. W., S.K., K.T., P.M.C., A.L., V.G., D.B., E.P.W., S.N., A.J.K.-S., J.A.D., A.J.B., K.-K.W., A.K.R.), F32CA174176 (K.A.W.), F30CA175133 (A.L.), T32 (CA115299-06 to A.L.); National Institute of Dental & Craniofacial Research (R21DE024396 to D.B. and H.N.); and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) R01DK114436 (H.N.), R01DK069984 (J.P.K.), R01DK101294 (J.P.K.), R01DK100342 (J.Q.), K01DK103953 (K.A.W.), T32DK007066 (K.A.W.) and P30-DK050306 Center of Molecular Studies in Digestive and Liver Diseases, The Molecular Pathology and Imaging, Molecular Biology/Gene Expression and Cell Culture Core Facilities; and K26RR032714 (H.N.) and P30-ES013508 University of Pennsylvania Center of Excellence in Environmental Toxicology (H.N.).Additional support was provided by American Cancer Society Grant (RP-10-033-01-CCE to A.K.R.), Fonds de Recherche en Santé du Québec (P-Giroux-27692) to V.G., NYSTEM (C029555) to J.Q. and Raptor Pharmaceuticals (H.N.).K.T. is a recipient of the Japan Society for the Promotion of Science Postdoctoral Fellowship. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Data availability.Microarray data were deposited at the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo)under accession number GSE37994.All data sets are available from the authors upon request. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "M.N., K.A.W., and H.N. designed and performed the majority of the experiments, analyzed the data, prepared the figures, and wrote the paper.M.N. generated and validated DNA constructs.M.N., S.K., K.T., P.M.C., and V.S. performed cell culture, transfection assays, retrovirus/lentivirus-mediated gene transfer, ChIP assays, flow cytometry, quantitative RT-PCR, immunoblotting, and mouse xenograft experiments.K.T., K.A.W., A.L., and V.G. performed 4NQO-induced carcinogenesis experiments.K. T., V.G., and K.A.W. generated and analyzed murine esophageal organoids.K.T., S.N., and K.A.W. performed IHC and IF.J.Q. and D.S.D. developed, validated, and provided K5Cre ERT2 ;p53 loxP/loxP mice and anti-ZEB1 antibody, respectively.Y.Y. and J.P.K. carried out DNA sequencing of human tumor samples.D.B., Y.K., and S.N. provided human tumor samples and analyzed clinical databases.A.J.K.-S.and S.N. reviewed and scored all immunohistochemistry samples.J.A.D., A.J.B., K.-K.W., and A.K.R. supervised the project and reviewed and edited the paper.E.P.W. performed biostatistics in experimental design and data analyses. Supplementary Information accompanies this paper at doi:10.1038/s41467-017-01500-9. Competing interests: The authors declare no competing financial 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": "M.N., K.A.W., and H.N. designed and performed the majority of the experiments, analyzed the data, prepared the figures, and wrote the paper.M.N. generated and validated DNA constructs.M.N., S.K., K.T., P.M.C., and V.S. performed cell culture, transfection assays, retrovirus/lentivirus-mediated gene transfer, ChIP assays, flow cytometry, quantitative RT-PCR, immunoblotting, and mouse xenograft experiments.K.T., K.A.W., A.L., and V.G. performed 4NQO-induced carcinogenesis experiments.K. T., V.G., and K.A.W. generated and analyzed murine esophageal organoids.K.T., S.N., and K.A.W. performed IHC and IF.J.Q. and D.S.D. developed, validated, and provided K5Cre ERT2 ;p53 loxP/loxP mice and anti-ZEB1 antibody, respectively.Y.Y. and J.P.K. carried out DNA sequencing of human tumor samples.D.B., Y.K., and S.N. provided human tumor samples and analyzed clinical databases.A.J.K.-S.and S.N. reviewed and scored all immunohistochemistry samples.J.A.D., A.J.B., K.-K.W., and A.K.R. supervised the project and reviewed and edited the paper.E.P.W. performed biostatistics in experimental design and data analyses. ",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "Supplementary Information accompanies this paper at doi:10.1038/s41467-017-01500-9. Competing interests: The authors declare no competing financial 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
}
] |
10.1186/1746-6148-9-130
|
HES1, a target of Notch signaling, is elevated in canine osteosarcoma, but reduced in the most aggressive tumors
|
<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>Hairy and enhancer of split 1 (HES1), a basic helix-loop-helix transcriptional repressor, is a downstream target of Notch signaling. Notch signaling and HES1 expression have been linked to growth and survival in a variety of human cancer types and have been associated with increased metastasis and invasiveness in human osteosarcoma cell lines. Osteosarcoma (OSA) is an aggressive cancer demonstrating both high metastatic rate and chemotherapeutic resistance. The current study examined expression of Notch signaling mediators in primary canine OSA tumors and canine and human osteosarcoma cell lines to assess their role in OSA development and progression.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>Reverse transcriptase - quantitative PCR (RT-qPCR) was utilized to quantify <jats:italic>HES1, HEY1, NOTCH1 and NOTCH2</jats:italic> gene expression in matched tumor and normal metaphyseal bone samples taken from dogs treated for appendicular OSA at the Colorado State University Veterinary Teaching Hospital. Gene expression was also assessed in tumors from dogs with a disease free interval (DFI) of <100 days compared to those with a DFI > 300 days following treatment with surgical amputation followed by standard chemotherapy. Immunohistochemistry was performed to confirm expression of HES1. Data from RT-qPCR and immunohistochemical (IHC) experiments were analyzed using REST2009 software and survival analysis based on IHC expression employed the Kaplan-Meier method and log rank analysis. Unbiased clustered images were generated from gene array analysis data for Notch/HES1 associated genes.</jats:p> <jats:p>Gene array analysis of Notch/HES1 associated genes suggested alterations in the Notch signaling pathway may contribute to the development of canine OSA. <jats:italic>HES1</jats:italic> mRNA expression was elevated in tumor samples relative to normal bone, but decreased in tumor samples from dogs with a DFI < 100 days relative to those with a DFI > 300 days. <jats:italic>NOTCH2 and HEY1</jats:italic> mRNA expression was also elevated in tumors relative to normal bone, but was not differentially expressed between the DFI tumor groups. Survival analysis confirmed an association between decreased HES1 immunosignal and shorter DFI.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p>Our findings suggest that activation of Notch signaling occurs and may contribute to the development of canine OSA. However, association of low HES1 expression and shorter DFI suggests that mechanisms that do not alter HES1 expression may drive the most aggressive tumors.</jats:p> </jats:sec>
|
[
{
"section_content": "Osteosarcoma (OSA) is the most common malignant bone tumor among children and adolescents with an incidence of 4.4 cases per million per year in the United States [1].OSA is also the most common spontaneous primary bone tumor of dogs, estimated to affect greater than 8,000 dogs annually in the United States [2].Tumor morphology, biological behavior, progression of disease and molecular characteristics are very similar in dogs and humans [2][3][4][5][6][7].Consequently, dogs provide a valuable comparative model of human OSA.Standard of care therapy for both human and canine OSA patients remains a combination of surgery and chemotherapy, with five-year survival rates reported in humans as high as 70% [1,8] and median survival in canine patients around 200 days [2].Unfortunately, in both human and canine patients approximately 80% are estimated to have micrometastases at presentation, some of whose tumors are also refractory to chemotherapy [2,8].These patients continue to have a poor prognosis.Histologic classification alone has not proven clinically relevant for determination of tumors likely to metastasize or exhibit resistance to chemotherapy protocols.The focus of recent research, therefore, has turned toward molecular characterization of primary tumors, especially aberrant gene and/or protein expression that might correlate with prognosis or chemotherapy sensitivity. Hairy and enhancer of split 1 (HES1), a basic helix-loophelix (bHLH) transcriptional repressor, is a downstream target of the Notch signaling pathway.The intracellular domain of activated Notch receptors (NICD) translocates to the nucleus, forms a transcriptional activating complex with recombination signal binding protein for immunoglobulin kappa J region (RPBJκ) and activates expression of target genes including HES1 [9,10].The HES1 protein contains both DNA-binding and protein-protein interaction domains important for its function as a transcriptional regulator (including negative regulation of its own transcription) [9,11,12].Notch-independent HES1 expression can also result from Hedgehog and c-Jun N-terminal kinase (JNK) signaling as well as from RAS/MAPK signaling [10,[13][14][15].Regulation of HES1 expression and activity is dependent on the tissue, spatial and temporal factors, and the proteins with which it interacts [9,10]. Overexpression of Notch and/or HES1 is associated with a variety of human cancers including T-cell acute lymphoblastic leukemia (ALL), and ovarian, breast, cervical, prostate, colon and non-small cell lung cancers [16][17][18][19].Notch/HES1 has also been shown to have tumor suppressor activity in some cancers including hepatocellular carcinoma, B-cell ALL, myeloid leukemia and neuroblastoma [20][21][22][23].In human OSA, Notch is implicated in OSA cell proliferation, invasion and metastasis [24,25].Increased HES1 mRNA expression was shown in some human OSA cells and OSA tumor samples compared to osteoblasts or normal bone and an association between high HES1 expression and decreased survival of OSA patients has been suggested [24][25][26][27].Reduced invasiveness in response to suppression of Notch signaling and HES1 activity implicates Notch/HES1 signaling in metastasis [28].Another study suggests both up-regulation of Notch and increased expression of HES1 in one OSA cell line occurs in response to activation of the Wnt/βcatenin pathway [29]. During bone development there is significant cross talk between the Wnt/β-catenin, hedgehog, and Notch pathways affecting osteoblast differentiation and maturation and influencing HES1 expression [10,[29][30][31].Like Notch and Wnt/β-catenin, aberrant hedgehog signaling is also associated with development of human cancers [31].Previous studies in our lab identified decreased expression of three hedgehog pathway associated genes in OSA tumors from dogs with a disease free interval (DFI) < 100 days (poor-responders) compared with tumors from dogs with a DFI > 300 (goodresponders) [32]. In order to explore the hypothesis that Notch signaling would be altered in canine OSA compared to normal bone samples, the current study examines the expression of NOTCH1 and 2 receptors and signaling targets, HES1 and HEY1, in canine OSA samples from patients with known outcome and normal bone tissues.Immunohistochemical analysis of HES1 protein was assessed in Kaplan-Meyer survival analysis to confirm the association of decreased HES1 expression with a shorter DFI. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Chemotherapy-naïve primary tumor samples were selected from the Colorado State University (CSU) Flint Animal Cancer Center's tissue archive.Samples are archived with owner consent and approval by the CSU Institutional Animal Care and Use Committee.Twenty tumors from good-and poor-responders (n = 10 each group) were selected following the protocol previously published [32].Briefly, chemotherapy-naïve primary OSA samples were from dogs treated with surgical amputation followed by chemotherapy with doxorubicin and/or a platinum based drug (distribution of choice of drug was not significantly different between groups).All twenty dogs were free of thoracic metastases by radiographic analysis at diagnosis and follow up consisted of evaluation by clinical examinations including thoracic radiographs every 2-3 months after initial treatment.Disease free interval (DFI) was calculated from surgery until development of metastatic disease and samples were identified for cohorts of good responders (DFI > 300 days) and poor responders (DFI < 100 days) in order to flank the median DFI (200 days).Nine additional appendicular OSA tumor samples were collected from which matched normal metaphyseal bone was harvested from the same limb (at least one joint space away from the tumor) following amputation.These nine matched samples were collected at amputation as cases came in (convenience sample) and absence/presence of metastasis, post-operative treatment, and patient follow-up were less consistent in this population.Tumor and normal bone fragments collected at amputation were flash-frozen in liquid nitrogen and stored at -80°C.Tumor fragments were also fixed in 10% neutral buffered formalin for 24 hours with subsequent routine processing and paraffin embedding. Immunohistochemical HES1 expression was also assessed in a subset of canine appendicular OSA patients from a previously reported multi-institutional randomized prospective clinical trial [33].The study was approved by the Institutional Animal Care and Use Committees of the participating institutions.All dogs underwent amputation followed by 5 cycles of adjuvant doxorubicin, with or without an investigational matrix metalloprotease inhibitor.Inclusion/exclusion criteria, staging, and follow-up procedures were standardized and tumor tissues were processed as previously reported [33].Histologic grading (from 1 to 3) was performed by one author (BEP) utilizing a schema incorporating amount of matrix, percent necrosis, nuclear pleomorphism, nucleolar size/ number and mitosis score [33].Mitotic index was calculated by counting the number of mitotic figures per 10 random 400× fields. ",
"section_name": "Tumor donors",
"section_num": null
},
{
"section_content": "Canine cell lines used in this study were provided by Dr. Douglas Thamm; all cell lines were validated for species and genetic identity using short-tandem-repeat (STR) profiling as previously described [34].Human OSA cell lines were obtained from Dr. Douglas Thamm (MG63, SAOS-2, SJSA-1), Dr. Hue Luu (MG63.2),or purchased from ATCC (U2OS).The MG63.2 cell line is a metastatic sub-line of the MG63 line, obtained via serial passage of rare lung metastases from MG63 [35].All non-purchased cell lines were validated prior to use using STR profiling by the University of Colorado DNA Sequencing Shared Resource.Cells were cultured in C10 media (DMEM high glucose with 4 mM L-glutamine (Hyclone Laboratories, Inc.), 1 mM of sodium pyruvate, 2× MEM vitamins, 1× MEM non-essential amino acids, 1× antibiotic-antimycotic (100×: 10,000 IU/ml penicillin, 10,000 ug/ul streptomycin and 25ug/ml) (all additives from Mediatech, Inc.), and 10% fetal bovine serum (FBS) (Atlas Biologicals, Fort Collins, CO). ",
"section_name": "Cell culture",
"section_num": null
},
{
"section_content": "Total RNA was extracted from tumors and RT-qPCR was conducted as described previously [32].Briefly, samples were freeze-fractured, homogenized, extracted with Trizol reagent (Invitrogen, Carlsbad, CA) and purified with RNeasy clean up (Quiagen, Valencia, CA) following manufacturer's protocols.RNA was extracted from normal bone using the same protocol with an additional spin of 800× g at 4°C for 5 minutes following homogenization.The supernatant was carried forward through the Trizol protocol.Total RNA was extracted from human and canine OSA cells using the RNeasy Kit (Qiagen) per the manufacturer's protocol.RNA was quantified via spectrophotometry and bioanalyzed for integrity as described in O'Donaghue et al. [32] with samples used having a RNA integrity number of at least 8. Human adult osteoblast total RNA was purchased from CELL Applications, Inc. Reverse transcriptase PCR and quantitative real time PCR cDNA synthesis was completed using the QuantiTect Reverse Transcription Kit (Qiagen) with 1 or 3 μg input RNA.RT-qPCR of cDNA was run using iQ SYBR Green Supermix (Bio-Rad) and 25 ng equivalent RNA input in 25 μL reactions on a Stratagene Mx3000P instrument.Expression in canine cells and tissues was normalized to hypoxanthine phosphoribosyltransferase 1 (HPRT1) expression.HPRT1 was selected based on its consistent moderate expression in our sample sets in prior microarray and RT-qPCR analysis (see Additional file 1 and reference [32]) and its previous use as a canine reference gene [36].Consistent with current recommendations for the selection of reference genes and because no single reference gene exhibited unchanged expression between samples, expression in human OSA cells was normalized to the geometric mean of four reference genes; ribosomal protein S15 (RBS15), glyceraldehyde-3-dehydrogenase (GAPDH), 18S ribosomal RNA (18SrRNA) and HPRT1 [37].Primer sequences and efficiencies for all genes and the full sequence of the canine HES1 amplicon are listed in Additional file 2. Primers were designed using Primer-Blast based upon NCBI RefSeq mRNA sequences when available.Primers were designed to be intron spanning when possible and cross-checked for specificity via UCSC in silico PCR.Primers were further validated with standard curves to calculate efficiency, and dissociation curves as previously described [34].RT-qPCR products were validated for size by agarose gel electrophoresis and sequenced to confirm identity.The 161 bp canine HES1 amplicon revealed 98% homology to the human homolog of HES1.Human HES1 primers used were the same as those used by Zhang et al. [24].The identity of the 200 bp amplicon was verified as human HES1 by dideoxy sequencing (CSU DNA sequencing Core). ",
"section_name": "RNA extraction",
"section_num": null
},
{
"section_content": "Western blot analysis was performed on canine and human OSA cells using whole cell lysates or cytoplasmic and nuclear fractions.Whole cell lysates were prepared in triethanolamine (TEA) lysis buffer (55 mM TEA, pH 7.5, 111 mM NaCl, and 2.2 mM EDTA, 0.44% SDS) with 1× Complete Protease Inhibitor Cocktail (Roche Diagnostics).Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay (Thermo Scientific).Nuclear extracts were prepared using a hypotonic 0.5% or 0.25% IgePal (NP-40) buffer (10 mM Hepes, 1.5 mM MgCl, and 10 mM KCl).Briefly, harvested cell pellets were re-suspended in IgePal buffer with protease inhibitor while vortexing, incubated on ice for 0-5 minutes, and centrifuged for 5 minutes at 500× g.The supernatant (cytoplasmic fraction) was collected and the pellet (nuclear fraction) was resuspended in TEA lysis buffer with protease inhibitors.Samples were separated using SDS-PAGE and transferred to a polyvinylidine fluoride membrane.The membrane was blocked with 5% non-fat dry milk (NFDM) for one hour at room temperature and incubated with rabbit monoclonal anti-HES1 antibody (RabMAb EPR4226, 1:500; Epitomics) in 5% bovine serum albumin (BSA) at 4°C overnight.After washing in 0.1% Tween 20-Tris-buffered saline (TBST) the membrane was incubated with secondary horseradish peroxidase conjugated goat anti-rabbit antibody (1:5000; Bio-Rad) in 5% NFDM for one hour at room temperature.SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology) was used to detect chemiluminescent signals.Band intensity from four experiments using whole cell lysates from MG63 and MG63.2 cell lines were analyzed using ImageJ software.The intensity of the HES1 band was normalized to the corresponding α-tubulin loading control. ",
"section_name": "Western blot",
"section_num": null
},
{
"section_content": "IHC to detect HES1 expression was performed on 4 μm sections from formalin-fixed paraffin embedded (FFPE) tumor tissues using standard immunoperoxidase techniques on charged slides with hematoxylin counter stain.Slides with sections were heated at 60°C for 30 minutes, allowed to cool, and deparaffinized with xylene or a citrus based clearing solution (Thermo-Fisher Scientific), and rehydrated with descending ethanol concentrations in deionized water (100%, 95%, 75% and 50%).Heat induced epitope retrieval was done with 10 mM sodium citrate buffer (pH 6.0) heated in a pressure cooker for 1 minute at 125°C.Endogenous peroxidase activity was blocked with 3% hydrogen peroxide at room temperature for 5 minutes with 3 washes in TBST both before and after.Slides were incubated with a non-serum protein block (Background Sniper, Biocare Medical) at room temperature for 15 minutes followed by incubation with primary antibody overnight at 4°C overnight.The primary antibody (anti-HES1 RabMAb, Epitomics) was used at a dilution of 1:750 (diluted in Antibody Diluent, Dako).Sections were then incubated with a prediluted secondary antibody conjugated to horseradish peroxidase (Envision and Dual Link System HRP, Dako) for 30 minutes at room temperature with 3 TBST washes both before and after.Diaminobenzidine (DAB, Ventana Medical Systems) was used as a chromogen for immunoreactive complex detection and slides were counterstained with hematoxylin. Sixty-one additional FFPE tumor samples were analyzed for HES1 immunohistochemical expression utilizing a protocol similar to that described above with the following exceptions: primary antibody was diluted in 2.5% normal goat serum in TBST (1:750 or 1:375, higher antibody concentration was used in subsequent batches to increase immunoreactivity signal), and detection was performed using biotinylated anti-rabbit IgG antibody in a Vectastain ABC Kit (Vector Laboratories).The IHC was performed in five batches of 8 to 18 slides each with the same antibody dilution used for an entire batch.Variations in antibody dilutions were controlled for by inclusion of a positive control tumor slide with a total immunoreactivity score of 4 (percent cells staining score of 2 and intensity score of 2; Table 1).All samples within each batch were scored in reference to the control.Negative controls lacking primary antibody were included in each batch. HES1 antibody validation was done using human placenta and canine lung and pancreas as positive control tissues.Specificity of the primary antibody was verified using a HES1 blocking peptide (Epitomics).Briefly, primary antibody was incubated with 25× (by mass) blocking peptide in antibody diluents (at both 1:375 and 1:750) for one hour at room temperature before application to canine control and sample tumor slides.Positive and negative controls with sections from the same tissues were incubated in parallel. Immunohistochemical scoring of all slides was performed independently by two authors blinded to case information.A positive cell was any neoplastic cell with distinct brown staining in the nucleus (stromal cells and endothelial cells were not counted).The percentage of positive cells in each sample was estimated based on an average of two or more high powered fields and scored as follows, 1: < 50% cells stain positive, 2: 50-75% cells stain positive, 3: > 75% cells stain positive.Average stain intensity ranged from 1 to 3 (lowest to highest intensity).Field location and number were selected randomly at the discretion of the individual scorer.The product of the percentage and intensity scores made up the overall immunoreactivity score (ranging from 1 to 9).Both scorers simultaneously reviewed slides with conflicting scores (scores deviating by more than 1 in either category) (n = 5) and consensus was reached.After review, total scores were averaged for statistical analyses. ",
"section_name": "Immunohistochemistry (IHC)",
"section_num": null
},
{
"section_content": "Immunocytochemistry was performed utilizing the same reagents and a similar protocol to that used for IHC.Slides were prepared via cytospin and dried overnight.Prior to the blocking step cells were fixed with 100% methanol at room temperature for 15 minutes, allowed to dry, washed in TTBS and incubated in 0.1% TritonX-100 in TBS for 7-12 minutes.The remainder of the procedure was identical to that used for IHC, but a higher concentration of primary antibody (1:250) was used. Photomicrographs (IHC and ICC) were taken using the Olympus BX51 Research System Microscope with an Olympus dp70 Digital Camera System.Minimal additional editing was done in Microsoft ® PowerPoint ® for Mac 2011. ",
"section_name": "Immunocytochemistry (ICC)",
"section_num": null
},
{
"section_content": "Total RNA from primary OSA tumor samples from dogs with DFI < 100 (n = 8) and DFI > 300 (n = 7) was analyzed on GeneChip Canine 2.0 Genome Arrays (Affymetrix, Santa Clara, CA) at CSU's Rocky Mountain Regional Center for Excellence (RMRCE) Genomics Core per Affymetrix protocols as described [35].Normal bone samples (n = 8) were analyzed using an identical protocol.Samples used for microarray analysis were a subset of those used for RT-qPCR (microarray samples were limited due to array costs).Microarray pre-processing combining the osteosarcoma samples with the normal bone samples was conducted using Probe Logarithmic Intensity Error (PLIER) estimation algorithms with log 2 transformations.Probesets including Notch receptor ligands, effectors, or targets of either the canonical Notch pathway or HES1 were selected based on literature review, Ingenuity® Systems Pathway analysis, and/or inclusion in The Human Notch Signaling Pathway RT 2 Profiler™ PCR Array (SAbiosciences) (Additional file 1).CIMminer was used to generate clustered images of the data from the 75 selected probesets with unsupervised clustering on both axes and the following parameters: average linkage, Euclidean distance, and quantile binning with median centering of the data.Full microarray data for the DFI groups is available through NCBI's Gene Expression Omnibus (GEO) via accession number GSE24251. ",
"section_name": "Gene expression microarray analysis",
"section_num": null
},
{
"section_content": "Statistical analysis of RT-qPCR and immunohistochemistry data (not including survival data) was performed using Prism software (GraphPad Software, La Jolla, CA).For RT-qPCR data standard curves, dissociation curves and amplification data was collected on a Stratagene Mx3000P instrument and analyzed using the Rest2009 software [38].HES1 RT-qPCR data was also analyzed using the 2 (-ΔΔCt) method [39] with similar results.IHC scores for the DFI > 300 and DFI < 100 tumors were analyzed with a 2-tailed Fischer's exact test after separating scores into low expression (total score less than 4) and high expression (total score greater than or equal to 4) categories.The cut off was based on results of receiveroperating characteristic (ROC) analysis of immunohistochemical scores for the DFI > 300 and DFI < 100 groups.Welch t-test in ArrayTrack 3.5.0with false discovery rate correction for multiple comparisons (FDR; based on all array probesets) was used to compare microarray gene expression data.Significance was defined as p < 0.05 (Welch t-test) or q < 0.05 (FDR).Statistical analysis of survival data was performed using a combination of Prism and SPSS software version 20 for Macintosh (IBM, Armonk, NY).Correlations between HES1 expression levels and other markers on a continuous scale were evaluated using linear regression analysis.A 2-tailed, unpaired t-test was used to evaluate the association between HES1 expression levels and categorical markers.The median DFI was estimated using the Kaplan-Meier method, and comparisons between groups made using log rank analysis for categorical variables.For continuous variables, markers were categorized into a low and high group using the median value as the break point.Multivariable Cox regression analysis was then performed, utilizing both forward and backward stepwise models.Variables identified with a univariate p-value of <0.1 were included in the multivariate analysis.For all other tests, p-values of <0.05 were considered significant. ",
"section_name": "Statistics",
"section_num": null
},
{
"section_content": "Gene expression analysis of Notch/HES1-associated genes groups normal and OSA bone samples, but does not distinguish DFI groups To assess the biological relevance of Notch/HES1 signaling in canine osteosarcoma, probesets including Notch receptor ligands, effectors, or targets of either the canonical Notch pathway or HES1 were selected from Canine 2.0 gene array data and analyzed for differential gene expression as described in materials and methods.Unbiased cluster analysis of data for the 51 Notch/ HES1-associated genes separated normal bone from tumors, but did not discriminate between the DFI groups (Figure 1).In total, 30 of 51 (58.8%)Notch/HES1 pathway associated genes examined were significantly different between tumor and normal bone (p < 0.05, q < 0.05); 23/30 (76.7%) had increased expression in tumors.Specifically, mRNA expression of NOTCH1 and NOTCH2 was elevated in tumor samples compared to normal bone (p < 0.05, q < 0.05).None of the genes evaluated had significantly different expression between DFI groups when corrected for multiple comparisons.HES1 was not included on the Canine 2.0 chip, but HEY1, another Notch target, was also elevated in tumors compared to normal bone (p < 0.05, q < 0.05). RT-qPCR analysis for NOTCH1, NOTCH2, HEY1 and HES1 was conducted on the normal bone/matched OSA and DFI tumor sample sets (Figures 2 and3).NOTCH1 exhibited decreased expression in the DFI < 100 day group relative to normal bone (FC down -1.656, p < 0.001), with no other significant changes measured.This result differed from the 1.27 fold upregulation of NOTCH1 identified in the gene array analysis, however previous studies have shown that fold-change differences <1.5 are frequently unreliable [40].Consistent with the array data, NOTCH2 exhibited an approximate 4-fold elevation in expression in both sets of DFI tumors, separately and in combination, relative to normal bone (p < 0.001).Similarly, HEY1 expression was elevated in each tumor group by a fold-change ranging from 6 to 10.2 (p ≤ 0.001).RT-qPCR analysis of these Notch signaling pathway elements confirmed our finding that Notch signaling is elevated in tumors relative to normal bone, but not between tumors in the two DFI groups. ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "RT-qPCR was also used to assess HES1 mRNA levels in OSA tumor and matched normal bone samples.Average HES1 mRNA expression was elevated 2.57-fold in canine OSA tumors compared to the matched normal bone (Figure 3A; p = 0.012); however, this fold change was highly variable when each OSA tumor was compared to its matched normal bone sample, with 5 tumors exhibiting elevated expression compared to normal bone and 4 tumors having virtually unchanged expression (Figure 3B, range 1.19-6.17-fold). We also assessed mRNA levels for HES1 in tumors taken from dogs with a DFI <100 days or DFI >300 days following treatment by amputation and chemotherapy.We found that HES1 expression was elevated 4.608-fold in the DFI > 300 tumors compared to the DFI < 100 group (Figure 3A; p < 0.001).HES1 expression in the DFI < 100 group was not different from the normal bone samples. Messenger RNA levels of HES1 were measured in canine and human osteosarcoma cell lines and confirmed using Western blot analysis using a rabbit monoclonal anti-human HES1 antibody as described to determine if HES1 mRNA levels correlated to protein expression, (Figures 4 and5, Additional file 3).Comparison of canine and human amino acid sequence of the HES1 gene identified 86% homology in the epitope targeted by this antibody.This was based on the predicted amino acid sequence of NCBI reference sequence XM_548669.1,which has been removed as a result of standard genome annotation processing.No additional canine HES1 record is currently available.Western blot analysis of whole cell OSA cell lysates revealed a 30 kD protein (HES1) as well as larger non-specific bands (Figure 4A,W).Given the role of HES1 as a transcriptional regulator, we hypothesized that active HES1 protein would reside in the nucleus.Western blot analysis of isolated nuclear and cytoplasmic fractions from both canine and human OSA cell lines confirmed enrichment of the 30 kD HES-1 protein in the nuclear fraction (Figure 4A, N) while the non-specific bands were enriched in the cytoplasm fraction (Figure 4A,C).Since equal amounts of total protein were loaded in each lane, the increased intensity and/or number of nonspecific bands in the cytoplasmic fraction were likely the result of concentration of these cytoplasmic proteins relative to total protein.Experiments using human OSA cells showed similar results (Additional file 3). HES1 mRNA and protein expression varied between cell lines in both canine and human OSA cells (Figure 5). For human cell lines mRNA expression was similar to that previously published [24,25].In general, HES1 mRNA expression was increased in canine cell lines relative to normal canine bone tissue (Figure 5A) and in human OSA cell lines relative to human osteoblasts (Figure 5C).Western blot analysis showed a characteristic band at 30 kDa with variable expression between cell lines (Figure 5B and5D).Interestingly, the metastatic subline of MG63 cells, MG63.2, exhibited elevated levels of mRNA compared to the MG63 line, but protein expression was not significantly different between the two lines (Additional file 4). We validated immunoreactivity using FFPE human placenta and found positive strong nuclear and cytoplasmic staining of placental macrophages (Hafbauer cells), moderate nuclear +/-cytoplasmic staining of stromal cells and light nuclear staining of endothelial cells consistent with Notch activity in placenta reported by Herr A B et al. [41].Staining of additional canine control tissues revealed positive punctate to diffuse intranuclear staining of pancreatic cells, endothelial cells and subsets of pulmonary epithelial cells as described in human literature [42][43][44] (see Additional file 5).Addition of a blocking peptide specific for the epitope targeted by our antibody eliminated all staining (data not shown).Immunocytochemistry of canine OSA cells (Gracie) showed diffuse nuclear staining consistent with the specific 30 kDa protein identified in the nuclear lysate by western analysis (Figure 4B). ",
"section_name": "HES1 mRNA expression in tumors and its prognostic significance",
"section_num": null
},
{
"section_content": "Once we established that the RabMAb anti-human HES1 antibody provided specific targeting of HES1 protein in human cultured cells and FFPE tissues with good cross-reactivity in canine samples, we performed immunohistochemistry using canine primary OSA samples.Of the 20 tumor samples from the canine DFI > 300 and DFI < 100 tumor groups, 14 were scored as described in the methods (Figure 6).For six samples, IHC was not possible due to loss of tissue during processing or poor quality/quantity of staining/tissue present.All OSA samples evaluated with immunohistochemistry had variable positive staining for HES1 both across tumors and within tumors.The staining pattern of tumor cells was predominantly nuclear with diffuse cytoplasmic staining less common.The median HES1 reactivity score was 3 (range, 1 to 9).Of the 6 tumors from dogs with DFI > 300 days, 83.3% (n = 5) had a score of greater than 3, compared to only 25.0% (n = 2) of the 8 tumors from dogs with DFI < 100 days (Table 1).Consistent with our RT-qPCR results, average HES1 immunohistochemical staining was lower in tumors from dogs with DFI < 100 days, but because of low power did not reach statistical significance (Additional file 6). To further assess the utility of HES1 protein expression as a prognostic biomarker, we performed IHC on 61 primary canine OSA tissues from a subset of dogs in a previously reported prospective clinical trial [33].Demographic information for this patient population is supplied in Additional file 7. IHC scores were assigned as described in materials and methods.HES1 was expressed in all tumors with a median HES1 immunoreactivity score of 4 in this population (range, 1 to 9).The overall median DFI was 168 (range 43 to 1,393+ days).The median DFI in dogs with a high HES1 immunoreactivity score (≥ 4) was 258 days compared to 155 days in dogs with a low HES1 immunoreactivity score (< 4) (p = 0.0023; Figure 7).Univariate analysis identified HES1, bone-specific alkaline phosphatase (BALP) activity, histologic grade, percent necrosis and mitotic index as potential predictors of DFI (Table 2, p < 0.1).Upon multivariate analysis, HES1, percent necrosis and mitotic index retained statistical significance (p = 0.029, 0.002 and 0.005 respectively; Table 2) as independent predictors of DFI.In summary, consistent with our prior RT-qPCR analysis, increased HES1 expression was identified as an independent prognostic biomarker for increased disease free survival in 61 canine OSAs treated by amputation and chemotherapy. ",
"section_name": "Increased immunohistochemical HES1 staining is associated with increased disease free interval",
"section_num": null
},
{
"section_content": "Expression of HES1 mRNA is frequently utilized as an indicator of Notch activity and Notch/HES1 activation has been implicated in a variety of human cancers with oncogenic activity in some tumor types and tumor suppressor activity in others [17][18][19][20][24][25][26][27].The goals of this study were to evaluate expression of Notch receptors and signaling mediators, HES1 and HEY1, in canine OSA samples from dogs with DFI > 300 days and DFI < 100 days as well as samples of matched OSA and normal bone to explore associations with OSA progression and patient outcome.Gene array analysis focusing on 51 Notch/HES1 associated genes identified elevated expression of Notch signaling mediators in tumors relative to normal bone.We confirmed a statistically significant elevation of NOTCH2, HEY1, and HES1 mRNA expression in OSA when compared with normal bone.Interestingly, we did not find elevated HES1 expression in the most aggressive OSA when comparing good and poor responders, but instead identified a statistically significant association between high HES1 mRNA and protein expression and longer DFI following standard treatment.Further, the gene array analysis of Notch/HES1 associated genes and RT-qPCR analysis of NOTCH1, NOTCH2 and HEY1 showed no significant differences in expression between the DFI groups.Overall, our findings indicate that alterations in Notch signaling occur during the development of canine OSA, but mechanisms that do not alter HES1 expression may drive the most aggressive tumors.The oncogenic role of Notch signaling in OSA in humans is supported by previous studies [24][25][26]; however, the specific role of HES1 is less clear.A common finding regarding HES1 expression between these previous studies and ours is the variability of expression within human and canine OSA cells and tumors (please note for references 24 and 28, that data from experiments done using the OS187 or COL cell lines should be viewed with caution due to a recent disclosure that these cells are not OSA cells) [24][25][26]28].For example, HES1 mRNA expression in tumors relative to normal bone was elevated in 5 of 9 canine tumors relative to matched normal bone samples in our study (Figure 3B) and 6 of 10 human tumors in the Tanaka study [25].There is also disagreement among studies as to which Notch receptors and target genes are functionally significant in OSA.Zhang et al. provided evidence that increased Notch1 activity and Notch1-induced expression of HES1 specifically are associated with invasion and metastasis in two OSA cell lines, the low HES1 expressing SAOS2 parental line and the metastatic, high HES1 expressing LM7 sub-line [24].Inhibition of Notch signaling by a gamma-secretase inhibitor suppressed LM7 OSA cell invasion, but had no effect on proliferation or tumorigenesis; whereas induced expression of intracellular cleaved Notch1 (ICN1) or HES1 in the SAOS2 line increased invasiveness.Tanaka et al. identified elevations of NOTCH2 and HEY1 mRNA in human OSA biopsy specimens relative to normal bone, but NOTCH1 and HES1 mRNA expression was not consistently elevated.In the same study, treatment of OSA cells and tumors grown in nude mice with a gamma-secretase inhibitor reduced proliferation through a G1 block [25].Differing results in these two studies may be due to different samples studied (tumor vs. cells) and/or the use of different gamma-secretase inhibitors.Our RT-qPCR data suggests that NOTCH2 and HEY1 may be primary mediators of Notch signaling in canine OSA as well.Interestingly, Zhang et al. observed both elevated HES1 mRNA expression [24] and elevated HES1 protein expression [28] in the LM7 metastatic sub-line relative to the SAOS2 parent line.We also observed an increase in HES1 mRNA expression in the MG63.2 metastatic sub-line relative to the MG63 parent line.However, western blot analysis identified similar levels of HES1 protein in the MG63 and MG63.2 lines suggesting that posttranscriptional regulation may be important.Studies exploring the relationship between HES1 expression and patient outcome in OSA are limited.Our RT-qPCR results (n = 20) revealed significantly increased HES1 mRNA expression in canine OSA from dogs with a longer DFI compared to those with a short DFI.This relationship was confirmed by immunohistochemical examination of HES1 protein in a larger dataset (n = 61).These results conflict with those of Hughes who conducted a RT-qPCR study using tissue from 16 primary OSAs that suggested lower HES1 mRNA expression may be associated with a better prognosis [27].Discrepancy from our results may be due to differing sample sizes, different measurements of outcome and different outcome groupings.Despite evidence of strong molecular similarities of canine and human OSA and high conservation of Notch/HES1 between species, there is also the possibility that canine tumors may exhibit different characteristics than their human counterparts.Until similar studies to evaluate nuclear immunoreactivity as a measure of protein expression are carried out in human tumors, no firm conclusions regarding possible differences in canine and human OSA with respect to HES1 expression can be made.Previous studies examining HES1 expression in other cancers or during development provide candidate mechanisms for reduced HES1 expression in the presence of elevated Notch signaling: uncoupling of HES1 from Notch signaling, cell cycle regulation of HES1 expression, and post-transcriptional regulation.HES1 expression has been reported to be uncoupled from Notch signaling in Ewing's sarcoma [15] and stimulation of HES1 transcription by sonic hedgehog (Shh) pathway occurs in mesodermal and neural stem cells [6 -8].Using RT-qPCR analysis, we identified significantly decreased SMO mRNA expression (p < 0.05) in the DFI < 100 tumors compared to the DFI > 300 tumors [32] suggesting that reduced HES1 expression in aggressive canine OSA might reflect a loss of Shh signaling.HES1 expression oscillations are both observed and necessary for cell cycle progression during neuronal development [45]; aggressive OSA tumor cells may utilize HES1 oscillatory patterns to manipulate the cell cycle and optimize their ability to metastasize and/or resist chemotherapy.Finally, several miRNAs have been shown to regulate HES1 (miR-124 and miR-23b) [46,47] and may contribute to altered HES1 expression in OSA cells and tumors. In addition, HES1 protein may exhibit specific functions depending on its phosphorylation status and binding partners.Kannan et.al. found that interactions with HES1 stimulates PARP1 activation and cleavage, ultimately resulting in apoptosis in B-ALL (overall a tumor suppressor role for HES1) [20].Further, in neuronal development, Ju et al. showed that HES1 interactions with phosphorylated PARP1 released HES1 from the HES1/ groucho/TLE repressor complex and, upon HES1 phosphorylation, led to association with a co-activator complex, changing the role of HES1 from a transcriptional repressor to a transcriptional activator [48].In bone development, via inhibition of RUNX2, Notch activity maintains a population of committed osteoblast precursors [49,50].Interestingly, several studies also show that HES1 binding stabilizes and activates RUNX2 protein; thus, HES1 has been shown to both inhibit and enhance the activity of RUNX2 [49,51].Additional studies exploring the phosphorylation status and binding partners of HES1 may provide a better understanding of these interactions in OSA. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "The results of the current study support the association of Notch pathway activation with the proliferative response of OSA.However, reduced HES1 expression in the most aggressive tumors despite the elevated expression of other Notch signaling effectors and targets indicates that HES1 is not an ideal sole surrogate marker of Notch signaling.Further, these findings suggest that additional mechanisms beyond Notch signaling may contribute to the aggressive phenotype of these tumors.Studies to define the role of Notch signaling in OSAs is warranted as inhibitors for this and other developmental pathways that impinge on HES1 are currently in clinical trials for the treatment of a variety of human cancers (summarized in Sang et al.) [52].Research in this area may reveal important regulatory mechanisms contributing to metastasis and therapeutic resistance in both canine and human OSA.While we found that HES1 expression was not consistently linked to Notch signaling in canine OSA, our study has determined that reduced HES1 expression serves as an independent prognostic biomarker. extraction from canine tissues and sectioning of FFPE canine tissues for IHC) and taught DDD and KPA RT-qPCR methodology including analysis of data.EJE provided guidance to DDD and JBC for IHC/ICC optimization and scoring.JBC assisted DDD with IHC and ICC optimization and scored IHC samples.TBB designed canine HES1 primers.DHT performed survival and regression statistical analyses.BEP graded histologic samples from the larger patient population.TJJ contributed to study design and provided canine HES1 primers.DLD conceived of the study design with TJJ, provided guidance and coordination for all experiments, and helped to draft the manuscript.All authors read and approved the final manuscript. ",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "We acknowledge Brian Kalet for extraction of RNA and protein lysates from human OSA cells as well as assistance troubleshooting various lab techniques and Todd Bass for technical advice/assistance for obtaining unstained tissue sections for IHC.This work was supported by grants to DLD from the Morris Animal Foundation (MAF D08CA-053).DDD was supported by NIH 9T32OD010437-11.KPA was supported by the Committee of Research and Ethics at the Norwegian School of Veterinary Science. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Additional file 1: Affymetrix Canine 2.0 microarray data processed with PLIER algorithm.Selected Notch signaling pathway genes from Affymetrix Canine 2.0 microarray data including both previously published [35] and unpublished data (normal bone). Additional file 2: Sequences, amplicon sizes, and efficiencies of primer pairs used in RT-qPCR experiments. Additional file 3: Western blot of MG63.2 and U20S whole cell, nuclear and cytosolic fractions for HES1.A distinct band at 30 kDa is present in both MG63.2 and U2OS human OSA whole cell (W) and is enriched in nuclear extract (N) lysates.Larger non-specific bands predominate in the cytoplasmic fraction (C).Equal amounts of total protein were loaded in each lane. Additional file 4: HES1 protein expression is not significantly different between MG63 and MG63.2 cell lines.HES1 band intensity normalized to α-tubulin loading control.Bars represent mean +/standard deviation from four independent experiments.Standard unpaired 2-tailed t-test was used to compare mean HES1 band intensity ratios for MG63 and MG63.2Western blot. Additional file 5: HES1 immunohistochemistry of control canine tissues.Variably intense nuclear staining is present in bronchiolar epithelial cells (A) and in both exocrine and endocrine (islets cells, blue circle) pancreatic cells (C).B and D are the negative controls.All photomicrographs were taken at 40× magnification; haematoxylin counterstain. Additional file 6: HES1 immunoreactivity in canine osteosarcomas from DFI < 100 and >300 groups.Immunoreactivity scores of nuclear HES1 protein expression in tumor sections from DFI < 100 day (filled circles, n = 8) and DFI > 300 day (filled squares, n = 6) groups.Horizontal line and error bars are mean ± SEM (p = 0.1026). Additional file 7: Summary demographic data for 61 canine patients from a previously reported clinical trial [33]. The authors declare that they have no competing interests. DDD carried out all mRNA and protein expression experiments (unless otherwise noted), scored IHC samples, analyzed data, performed statistical analyses (except for survival and regression analyses) and drafted the manuscript.KPA contributed to study design and carried out HES1 RT-qPCR for the DFI group tumors.LEP carried out sample preparation (RNA ",
"section_name": "Additional files",
"section_num": null
},
{
"section_content": "Additional file 1: Affymetrix Canine 2.0 microarray data processed with PLIER algorithm.Selected Notch signaling pathway genes from Affymetrix Canine 2.0 microarray data including both previously published [35] and unpublished data (normal bone). Additional file 2: Sequences, amplicon sizes, and efficiencies of primer pairs used in RT-qPCR experiments. Additional file 3: Western blot of MG63.2 and U20S whole cell, nuclear and cytosolic fractions for HES1.A distinct band at 30 kDa is present in both MG63.2 and U2OS human OSA whole cell (W) and is enriched in nuclear extract (N) lysates.Larger non-specific bands predominate in the cytoplasmic fraction (C).Equal amounts of total protein were loaded in each lane. Additional file 4: HES1 protein expression is not significantly different between MG63 and MG63.2 cell lines.HES1 band intensity normalized to α-tubulin loading control.Bars represent mean +/standard deviation from four independent experiments.Standard unpaired 2-tailed t-test was used to compare mean HES1 band intensity ratios for MG63 and MG63.2Western blot. Additional file 5: HES1 immunohistochemistry of control canine tissues.Variably intense nuclear staining is present in bronchiolar epithelial cells (A) and in both exocrine and endocrine (islets cells, blue circle) pancreatic cells (C).B and D are the negative controls.All photomicrographs were taken at 40× magnification; haematoxylin counterstain. Additional file 6: HES1 immunoreactivity in canine osteosarcomas from DFI < 100 and >300 groups.Immunoreactivity scores of nuclear HES1 protein expression in tumor sections from DFI < 100 day (filled circles, n = 8) and DFI > 300 day (filled squares, n = 6) groups.Horizontal line and error bars are mean ± SEM (p = 0.1026). Additional file 7: Summary demographic data for 61 canine patients from a previously reported clinical trial [33]. ",
"section_name": "Additional files",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests. ",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "DDD carried out all mRNA and protein expression experiments (unless otherwise noted), scored IHC samples, analyzed data, performed statistical analyses (except for survival and regression analyses) and drafted the manuscript.KPA contributed to study design and carried out HES1 RT-qPCR for the DFI group tumors.LEP carried out sample preparation (RNA ",
"section_name": "Authors' contributions",
"section_num": null
}
] |
10.1186/s10020-021-00413-0
|
Microfibrillar-associated protein 5 regulates osteogenic differentiation by modulating the Wnt/β-catenin and AMPK signaling pathways
|
<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>Dysfunctional osteogenesis of bone marrow mesenchymal stem cells (BMSCs) plays an important role in osteoporosis occurrence and development. However, the molecular mechanisms of osteogenic differentiation remain unclear. This study explored whether microfibrillar-associated protein 5 (MFAP5) regulated BMSCs osteogenic differentiation.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>We used shRNA or cDNA to knock down or overexpress MFAP5 in C3H10 and MC3T3-E1 cells. AR-S- and ALP-staining were performed to quantify cellular osteogenic differentiation. The mRNA levels of the classical osteogenic differentiation biomarkers Runx2, Col1α1, and OCN were quantified by qRT-PCR. Finally, we employed Western blotting to measure the levels of Wnt/β-catenin and AMPK signaling proteins.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>At days 0, 3, 7, and 14 after osteogenic induction, AR-S- and ALP-staining was lighter in MFAP5 knockdown compared to control cells, as were the levels of Runx2, Col1α1 and OCN. During osteogenesis, the levels of β-catenin, p-GSK-3β, AMPK, and p-AMPK were upregulated, while that of GSK-3β was downregulated, indicating that Wnt/β-catenin and AMPK signaling were activated. The relevant molecules were expressed at lower levels in the knockdown than control group; the opposite was seen for overexpressing cell lines.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>MFAP5 regulates osteogenesis via Wnt/β‑catenin- and AMPK-signaling; MFAP5 may serve as a therapeutic target in patients with osteoporosis.</jats:p> </jats:sec>
|
[
{
"section_content": "Osteoporosis is very common worldwide, and is associated with bone fragility caused by osteopenia, reduced bone mass, and an increased fracture risk (Coughlan and Dockery 2014;Kanis 1994;Wang et al. 2009). Osteoporosis affects motor function and is also associated with secondary complications, including fracture and skeletal deformities, which affect health and the quality of life (Black and Rosen 2016;Kanis 1994).Accidental trauma often triggers bone malformation or even non-healing, which are associated with poor prognosis (Hegde et al. 2016).It is accepted that reduced proliferation and osteogenesis of bone marrow mesenchymal stem cells (BMSCs) are closely associated with osteoporosis occurrence and progression (Liu et al. 2021;Luo et al. 2019).But due to the unclear mechanism of osteogenic differentiation, few methods could be applied to treat osteoporosis in clinical by targeting osteoblastic cells.More effective genes that could regulating osteogenesis need to be identified which could provide potential targets and develop drugs for patients suffering from osteoporosis. The Wnt/β-catenin signaling pathway plays an important role in osteogenic differentiation and bone regeneration (Chen et al. 2021;Leucht et al. 2019).When Wnts bind to the low density lipoprotein receptor-associated proteins (LRPs) and Frizzled transmembrane receptors, the classic Wnt/β-catenin signaling pathway would be activated (Siracusa et al. 2021).After that, the β-catenin migrates into the nucleus and regulates osteogenic gene transcription (Huybrechts et al. 2020).Previous study found that Morusin could promote osteogenic differentiation of BMSCs via the activation of Wnt/β-catenin signaling pathway (Chen et al. 2021).Similarly, vasoactive intestinal peptide (VIP) could also increase the BMSCs through activating the Wnt/β-catenin pathway (Shi et al. 2020).Recently, the AMPK signaling was found could positively regulate osteogenesis.Compared with wildtype and AMPK+/-mice, the AMPK-/-mice showed a retardation of postnatal bone development (Kanazawa et al. 2018).Metformin could directly contribute to osteogenesis by activating AMPK and expression of Runx2 (Molinuevo et al. 2010).All these findings suggested the crucial function of Wnt/β-catenin and AMPK pathways in regulating BMSCs osteogenesis. Microfibrillar-associated protein 5 (MFAP5) was a component of extracellular matrix (ECM) genes.The N-terminal of MFAP5 contains an Arg-Gly-Asp sequence (RGD) domain, which united with the elastic fibers in ECM and regulating the function of it (Deford et al. 2016).MFAP5 is crucial in regulating cell motility and signal transduction (Albig et al. 2008).Bioinformatics analyses indicate that, in osteoblasts, MFAP5 is highly expressed (Zhu et al. 2021).In the meanwhile, it is found that, during the process of osteoblastic differentiation, the MFAP5 has an increasing expression pattern with many other osteogenesis biomarkers, including Runx2, type I collagen, Msx-2, Dlx-5, etc. (Burns et al. 2010).But the role of MFAP5 in osteogenic differentiation is still not clear.Through analyzing bioinformatic data, we found that MFAP5 expression was downregulated in BMSCs in osteoporosis patients.In the meanwhile, during osteogenesis, MFAP5 expression tended to increase.We thus hypothesized that MFAP5 might regulate osteogenic differentiation.Through silencing or overexpressing MFAP5 in mouse osteoblastic C3H10 and MC3T3-E1 cells, the role of MFAP5 in regulating osteogenesis was tested.We stained cells for alkaline phosphatase (ALP) and Alizarin Red S (AR-S) and measured the expression levels of the osteogenic biomarkers Runx2, Col1α1, and OCN.It's shown that MFAP5 strongly promoted osteoblastic differentiation.Also, the expression patterns of key proteins in the Wnt/β-catenin and AMPK signaling pathways were affected by MFAP5 knockdown or overexpression.The results indicated that MFAP5 served as an osteogenic factor.This improves our understanding of bone metabolism; moreover, a potential therapeutic target for osteoporosis was identified. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "GSE156508 and GSE80614 gene expression profiles were obtained from the GEO database (https:// www.ncbi.nlm.nih.gov/).GSE156508 reflects the gene expression pattern of primary osteoblasts (OBs) in women with osteoporotic fractures or severe osteoarthritis; sequencing was performed using GPL16686 of the Human Gene 2.0 ST Array (Affymetrix, Santa Clara, CA, USA).GSE80614 reflects the gene expression pattern of osteogenically differentiated hMSCs at 0, 1, 2, 3, and 4 days.For sequencing, GPL6947 of the Human HT-12 V3.0 expression head chip was used (Illumina, San Diego, CA, USA).The data were analyzed using the GEO2R online tool. ",
"section_name": "Microarray data",
"section_num": null
},
{
"section_content": "AR-S (catalog no.A553), dimethyl sulfoxide (DMSO; D2650), ascorbic acid (AA; A4403), β-glycerophosphate (β-GP; G9422), and dexamethasone (DXMS; D4902) were purchased from Sigma-Aldrich (St. Louis, MO, USA).AR-S, AA, and β-GP were dissolved in phosphatebuffered saline (PBS) to concentrations of 40 mM (pH 4.2), 10 mM, and 1 M, and stored at 4 °C.DXMS was dissolved in DMSO (to 1 mM) and stored at -20 °C.The primary antibodies used were anti-MFAP5 (DF13146, Affinity, USA), -β-catenin (sc7199; Santa Cruz Biotechnology, Santa Cruz, CA, USA), -phospho-GSK-3β (5558; Cell Signaling Technology, Danvers, MA, USA), -GSK-3β (12456; Cell Signaling Technology), -AMPK (9158; Cell Signaling Technology), -phospho-AMPK (5759; Cell Signaling Technology), -Notch1 (4380; Cell Signaling Technology), and -GAPDH (G9295; Sigma).The secondary antibodies were goat anti-rabbit or -mouse antibodies (7074,4410;Cell Signaling Technology). ",
"section_name": "Reagents and antibodies",
"section_num": null
},
{
"section_content": "C3H10 and MC3T3-E1 cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and grown in high-glucose Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, NY, USA) or α-MEM (Gibco) with 10% (v/v) fetal bovine serum (FBS; Gibco) at 37 °C under 5% (v/v) CO 2 in a humid atmosphere.The effect of MFAP5 on proliferation of cells were tested by a Cell Counting Kit 8 (Dojindo, Kumamoto, Japan) according to the instructions.The osteogenic induction medium was 10 mM β-GP, 50 mM AA, and 100 mM DXMS in growth medium.The growth or induction medium was replaced every other day. ",
"section_name": "Cell culture, cell counting kit 8 assay and osteoblastic differentiation",
"section_num": null
},
{
"section_content": "The plasmids psPAX2, pMD2.G, and pLKO.1-EGFPpuromycin were purchased from GeneChem (Shanghai, China) and used to deliver lentivirus-expressing short hairpin RNA (shRNA).Three shRNAs (shRNA1: 5′-CCG GCG GGA TGA GAA GTT TGC TTG TCT CGA GAC AAG CAA ACT TCT CAT CCC GTT TTTTG-3′, shRNA2: 5'-AAA ACA CCA GTT TAC GAC GTA TGT ATT CGT ACA TAC GTC GTA AAC TGG TGC-3′, and shRNA3: 5′-CCG GGA GAT GAT GTG CCT GAG ACA TCT CGA GAT GTC TCA GGC ACA TCA TCT CTT TTTTG-3′) were used to knock down MFAP5 expression.The full-length DNA coding sequence of MFAP5 was amplified and inserted into the lentiviral vector pLKO.1-EGFP-puromycin to allow MFAP5 overexpression in target cells.The recombinant lentiviruses were used to infect cells at about 60% confluence for 2 days.Transfected cells were selected by growth in puromycin (Sigma) for 2 weeks. ",
"section_name": "Plasmid and viral infections",
"section_num": null
},
{
"section_content": "Seeded C3H10 and MC3T3-E1 cells were allowed to grow for 0, 3, 7, and 14 days.At each time point, the cells were rinsed twice in PBS and fixed in polyformaldehyde for 15 min at 37 °C.An ALP staining kit (DE0004; Leagene, Beijing, China) was used as instructed by the manufacturer.Cells were stained with an AR-S solution for 35 min at 37 °C.PBS was used to wash away excess stain.The stained cells were then photographed, with the exposure time and white balance held constant. ",
"section_name": "ALP and AR-S staining",
"section_num": null
},
{
"section_content": "AR-S, used for staining cells, was dissolved in 10% (w/v) cetylpyridinium chloride in PBS, and absorbance was measured at 562 nm.To measure ALP levels, stained cells were collected from plates and lysed in lysis buffer (Beyotime, Shanghai, China).The protein-containing supernatants were collected, and the total protein concentrations were normalized using the BCA method (23228; Thermo Scientific, Waltham, MA, USA).A test kit from Jiancheng Biotechnology (Nanjing, China) was used to measure ALP activities. ",
"section_name": "AR-S and ALP assays",
"section_num": null
},
{
"section_content": "Adherent cells at various time points were washed twice with cold PBS; 65 μL of RIPA buffer with 1% (w/v) phenylmethylsulfonyl fluoride (PMSF) was added to each 60-mm dish and the cells scraped into Eppendorf (EP) tubes.The lysates were placed on ice for 20 min with vortexing for 3 s every 5 min, and then centrifuged (10 min, 12,000 rpm, 4 °C); the supernatant protein levels were normalized using a BCA Protein Assay Kit (Beyotime) according to the manufacturer's instructions.The proteins were subjected to gel electrophoresis and transferred to poly (vinylidene fluoride) (PVDF) membranes (Millipore, Billerica, MA, USA).The membranes were blocked with skim milk and incubated for 15 h at 4 °C with primary antibodies.Goat anti-rabbit or -mouse secondary antibodies (7074, 4410; Cell Signaling Technology) were used to probe the membranes at room temperature for 2 h.An ECL kit (Zhejiang Share Bio, Zhejiang, China) was employed to detect bands, and images were obtained using the Fluor Chem E system (ProteinSimple, Santa Clara, CA, USA).ImageJ (1.8.0) for Windows was applied to quantify the results of western blotting. ",
"section_name": "Western blotting",
"section_num": null
},
{
"section_content": "Total cellular RNA was extracted into RNAiso Plus (9108; Takara, Shiga, Japan) according to the manufacturer's protocol.An Infinity 200-Pro multi-well plate reader (Tecan, Männedorf, Switzerland) was used to assess the concentrations and qualities of RNA samples.RNA was reverse-transcribed to cDNA using the PrimeScript RT Master Mix (RR036A; Takara).cDNA samples were mixed with SYBR Premix Ex Taq (RR420A; Takara), and forward and reverse primers for quantitative real-time PCR (qRT-RCR) performed as follows: 95 °C for 10 min followed by 95 °C for 10 s, 60 °C for 115 s, and 72 °C for 15 s (40 cycles).The data were analyzed using the 2 -△△Ct method.All qRT-PCR primers are listed in Table 1. ",
"section_name": "Quantitative real-time PCR",
"section_num": null
},
{
"section_content": "All experiments were repeated at least three times.The results were analyzed using GraphPad Prism for Windows (ver.8.0; GraphPad Software Inc., La Jolla, CA, USA) and are presented as means ± standard deviations.Groups were compared using the two-tailed Student's t-test; differences among more than two groups were analyzed by one-way ANOVA.P-values of < 0.05, < 0.01 and < 0.001 were considered statistically significant. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To explore the potential role played by MFAP5 in osteogenesis, we analyzed the GSE156508 database, which includes data on the primary osteoblasts of women with osteoporotic fractures (n = 6) and severe osteoarthritis (n = 6).MFAP5 expression was significantly decreased in the primary osteoblasts of the osteoporosis compared to osteoarthritis group (Fig. 1A).As the control group contained patients with osteoarthritis who are different from healthy individuals, data interpretation was not quite convincible.Thus, we obtained data on osteogenically differentiated hMSCs at 0, 1, 2, 3, and 4 days, and found that MFAP5 expression was positively correlated with osteogenesis (Fig. 1B).We then evaluated MFAP5 expression during osteogenesis of the mouse osteoblastic cell lines C3H10 and MC3T3-E1 (Fig. 1C,G,H).MFAP5 expression increased during osteogenesis in both protein and mRNA.We determined the extent of osteogenic differentiation by staining the cells and performing qRT-PCR of mRNAs encoding osteogenic biomarkers.As osteogenesis progressed, mineralization and osteogenesis were enhanced, and ALP and AR-S staining Table 1 The sequences of qRT-PCR primers became more intense (Fig. 1D-F).Also, the expression levels of the osteogenic biomarkers Runx2, Col1α1, and OCN increased (Fig. 1G andH), suggesting a positive correlation between MFAP5 expression and osteoblast differentiation. ",
"section_name": "MFAP5 expression correlated positively with osteogenesis",
"section_num": null
},
{
"section_content": "",
"section_name": "Gene",
"section_num": null
},
{
"section_content": "MFAP5-knockdown C3H10 and MC3T3-E1 cell lines were established using a lentivirus transfection system.Three shRNAs targeting MFAP5 were used.MFAP5 expression was evaluated in protein and mRNA.As shown in Fig. 2A and B, MFAP5 expression was significantly reduced in the MFAP5-shRNA3 C3H10 and MC3T3-E1 cell lines. ",
"section_name": "Establishment of MFAP5 knockdown cell lines",
"section_num": null
},
{
"section_content": "After establishing MFAP5 knockdown lines, we assessed the role played by MFAP5 in osteogenic differentiation. As shown in Fig. 2C, ALP and AR-S staining were used to qualitatively assess the extent of osteogenesis and mineralization in negative control (NC) and MFAP5-shRNA (shRNA) groups of C3H10 and MC3T3-E1 cells; dye staining was less intense in the two shRNA groups.The quantitative ALP levels and AR-S absorbance were consistent with the staining data (Fig. 2D andE).For the cell numbers would affect the degree of staining, the proliferation abilities of cells were tested and there were no significant differences (see Additional file 1).We extracted mRNAs at various times during osteogenesis; qRT-PCR indicated that the mRNA expression levels of the osteogenic biomarkers Runx2, Col1α1, and OCN were lower in the shRNA groups than the NC group (Fig. 2F-H).Thus, MFAP5 knockdown significantly inhibited osteogenic differentiation. ",
"section_name": "MFAP5 knockdown inhibited osteogenic differentiation",
"section_num": null
},
{
"section_content": "We explored how MFAP5 regulated osteogenesis using Western blotting to quantify key proteins in osteogenic differentiation-related signaling pathways in the NC and shRNA groups.Figure 3A shows that the β-catenin, p-GSK-3β, AMPK, and p-AMPK levels increased, while that of GSK-3β decreased, during osteogenic induction, indicating that the Wnt/β-catenin and AMPK signaling pathways were activated.Also, Wnt/β-catenin and AMPK signaling were inhibited in the shRNA groups compared to the control during osteogenic differentiation of both C3H10 and MC3T3-E1 cells.In the meanwhile, we also quantified the results of western blotting which were shown in Fig. 3B-D.The protein level of β-catenin was higher in the control groups in both two cells lines.The trends of p-GSK-3β /GSK-3β was consistent to the result of β-catenin.As for the AMPK signaling pathway, when the osteogenic differentiation started, the p-AMPK level sudden increased, and lead the increase of AMPK.Because there was a positive feedback relationship between p-AMPK and AMPK, there was no clear trend of the value of p-AMPK/AMPK.But, the expression of AMPK, p-AMPK and p-AMPK/AMPK are significantly higher in the control group which indicating that the AMPK signaling was suppressed in the shRNA group.Therefore, the Wnt/β-catenin and AMPK signaling pathways were active when MFAP5 promoted osteogenic differentiation. ",
"section_name": "MFAP5 knockdown inhibited Wnt/β-catenin and AMPK signaling",
"section_num": null
},
{
"section_content": "To confirm that MFAP5 regulated osteogenesis, we established MFAP5-overexpressing C3H10 cell lines.The MFAP5 sequence was amplified and transfected into cells, and MFAP5 expression was measured at both the protein and mRNA levels.As shown in Fig. 4A andB, compared to the controls, MFAP5 expression increased significantly.AR-S and ALP staining were more intense during osteogenesis, indicating greater mineralization and osteogenesis in MFAP5-overexpressing (from cDNA) cells (Fig. 4C).The ALP activity assay and qualitative AR-S staining intensity supported this conclusion (Fig. 4D andE).Protein quantification during osteogenesis indicated that MFAP5 overexpression activated the Wnt/β-catenin and AMPK signaling pathways (Fig. 4F-I).In summary, all of the data indicated that MFAP5 positively regulated osteogenic differentiation by activating Wnt/β-catenin and AMPK signaling The Runx2, Col1α1, and OCN levels were higher in the test cells than the control during osteogenesis (Fig. 4J-L). ",
"section_name": "Overexpression of MFAP5 promoted osteogenic differentiation and activated Wnt/β-catenin and AMPK signaling",
"section_num": null
},
{
"section_content": "Osteoporosis is a major public health problem worldwide; there is no satisfactory therapy (Bone et al. 2017;Eastell and Szulc 2017;Kanis 1994;Naylor et al. 2016).Dysfunctional osteogenic differentiation of BMSCs is associated with osteoporosis initiation and development, accompanied by a significant decline in osteogenic differentiation and increased adipogenic differentiation (Chen et al. 2016;Li et al. 2018).BMSCs can develop into cells of different types; the mechanism that determines the direction of differentiation remains unclear (Guo et al. 2020;Liu et al. 2015).A better understanding of BMSC osteogenesis is needed.In this study, we investigated the role of MFAP5 in regulating BMSCs osteogenic differentiation by activating the Wnt/β-catenin and AMPK signaling pathways.We examined the GEO database and found that MFAP5 expression in the BMSCs of osteoporosis patients was lower than in controls, implying that MFAP5 might play a role in BMSC osteogenic differentiation.By inducting the C3H10 mouse mesenchymal stem cell line and MC3T3-E1 mouse embryonic osteoblast progenitor cell line into osteoblasts, we found the expression of MFAP5 was upregulated during this process.After silencing it in these two cells lines, the osteogenic differentiation ability was declined.Many signaling pathways play essential roles in osteogenesis and bone formation.Previous studies have shown that the canonical Wnt/β-catenin signaling pathway positively regulates osteogenic differentiation (Hong et al. 2019;Wang et al. 2018).In normal cytoplasm atmosphere, the expression of glycogen synthase kinase 3β (GSK-3β) brakes the stability of β-catenin, inhibiting its further function of cellular metabolic regulation.When Wnt signaling is activated (activation this pathway lies on the cell membrane binding of Wnt and frizzled receptors and the LRP co-receptor), the degradation of GSK-3β is enhanced, eventually promoting β-catenin stabilization and nuclear translocation (Wang et al. 2018).Notably, the phosphorylated form of GSK-3β is degraded (Kim et al. 2017;Li et al. 2012;Oh et al. 2014).Based on the character of this signaling pathway, we detected the expression of p-GSK-3β, GSK-3β and β-catenin.We found that, during osteogenic differentiation, β-catenin and p-GSK-3β were upregulated, while GSK-3β was downregulated in the shRNA groups, indicating that Wnt/β-catenin signaling was suppressed after knocking down MFAP5. AMPK was recently shown to play a role in osteogenesis by promoting Runx2, ALP, and OCN (Kim et al. 2018;Wang et al. 2013;Wang et al. 2016).We found that the AMPK and p-AMPK levels gradually increased during osteogenesis, consistent with previous reports.However, the p-AMPK level fell after MFAP5 knockdown, suggesting that AMPK signaling was involved in the MFAP5 regulation of osteogenesis.Of note, MFAP5 activated Notch 1 signaling in certain tumors, promoting tumor invasion and migration (Chen et al. 2020;Li et al. 2019).Notch 1 signaling is also involved in osteogenesis (Díaz-Tocados et al. 2017;Fan et al. 2016;Fan et al. 2021).However, the Notch 1 levels of the MFAP5 knockdown and control groups did not differ significantly (see Additional file 2).Ann et al. found that Runx2 inhibited Notch 1 signaling (Ann et al. 2011).The Runx2 expression level of MFAP5-knockdown BMSCs was lower than that of control cells.The opposing effects of MFAP5 and Runx2 on Notch 1 signaling in BMSCs might cancel out their activities; this possibility should be studied further. The main finding of this study was that MFAP5 knockdown decreased the levels of β-catenin, phosphorylated GSK-3β, AMPK, and downstream osteogenic biomarkers, while MFAP5 overexpression had the opposite effects.Thus, MFAP5 regulates osteoblast differentiation via the Wnt/β-catenin and AMPK signaling pathways.Interestingly, Wnt/β-catenin and AMPK pathways were found significantly suppress adipogenic differentiation (Chen et al. 2014;Takada et al. 2009).Previous study found that MFAP5 is high expressed is adipose tissue (Vaittinen et al. 2011).But during the process of adipogenic differentiation, the MFAP5 expression significantly decreased (more than 80%) from the 6th day of adipogenesis (Vaittinen et al. 2015).This finding suggests MFAP5 might also participate in regulating adipogenic differentiation.Based on the results of this study, it may trigger a switch from adipogenesis to osteogenesis as BMSCs differentiate; we are currently investigating this possibility.Further studies on how MFAP5 regulates BMSC differentiation might identify other potential therapeutic targets, or useful small-molecule drugs.MFAP5 is small (25 kDa) and the recombinant protein is easy to synthesize.The protein is expressed mainly on microfibrils of the extracellular matrix.Thus, exogenous MFAP5 may be compatible with cell surfaces.Yeung et al. used an immunological approach to successfully block MFAP5; this enhanced the chemosensitivity of ovarian and pancreatic cancer (Yeung et al. 2019).Similar methods might be used to treat osteoporosis. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "We found that MFAP5 promoted osteogenic differentiation of MSCs by activating the Wnt/β-catenin and AMPK signaling pathways, which is a potential therapeutic target for bone metabolism diseases. • 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": "Conclusion",
"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": "Thanks to Zhenxu Wang from the Shanghai Children's Hospital for her enlightening advices of this research design. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This work was supported by the Key Department of Minhang District (2020MWTZB03), the Key Department of the Fifth People's Hospital of Shanghai (2020WYZDZK03), the Fifth People's Hospital of Shanghai, Fudan University (2018WYZT01), the Fifth People's Hospital of Shanghai, Fudan University (N123E5) and the Minhang District Leading Talent Development Funds. ",
"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": "BMSCs: Bone marrow stromal cells; MFAP5: Microfibrillar-associated protein 5; ALP: Alkaline phosphatase; AR-S: Alizarin red S; qRT-PCR: Quantitative real-time polymerase chain reaction; DXMS: Dexamethasone; AA: L-ascorbic acid; β-GP: β-Glycerophosphate; DMEM: Dulbecco's modified eagle's medium; FBS: Fetal bovine serum; DMSO: Dimethyl sulfoxide; shRNA: Short hairpin RNA; LRPs: Lipoprotein receptor-associated proteins; ECM: Extracellular matrix; NC: Negative control; Runx2: Runt-related transcription factor 2; Col1α1: Pro-alpha1 chains of type I collagen; OCN: Osteocalcin; GSK-3β: Glycogen synthase kinase 3β. The online version contains supplementary material available at https:// doi.org/ 10. 1186/ s10020-021-00413-0. Additional file 2. The expressions of Notch1 signaling in different groups. HRL and WLZ: The acquisition, analysis, interpretation of the data for the work.SWS and HTH: Substantial contributions to the conception and design of the work.TLZ: Revising this work critically for important intellectual content.MHW: Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.All authors read and approved the final manuscript. Ethics approval and consent to participate Not available. Not available. 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": "Abbreviations",
"section_num": null
},
{
"section_content": "BMSCs: Bone marrow stromal cells; MFAP5: Microfibrillar-associated protein 5; ALP: Alkaline phosphatase; AR-S: Alizarin red S; qRT-PCR: Quantitative real-time polymerase chain reaction; DXMS: Dexamethasone; AA: L-ascorbic acid; β-GP: β-Glycerophosphate; DMEM: Dulbecco's modified eagle's medium; FBS: Fetal bovine serum; DMSO: Dimethyl sulfoxide; shRNA: Short hairpin RNA; LRPs: Lipoprotein receptor-associated proteins; ECM: Extracellular matrix; NC: Negative control; Runx2: Runt-related transcription factor 2; Col1α1: Pro-alpha1 chains of type I collagen; OCN: Osteocalcin; GSK-3β: Glycogen synthase kinase 3β. ",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https:// doi.org/ 10. 1186/ s10020-021-00413-0. ",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "Additional file 2. The expressions of Notch1 signaling in different groups. ",
"section_name": "Additional file 1. Proliferation capacity of cells in different groups.",
"section_num": null
},
{
"section_content": "HRL and WLZ: The acquisition, analysis, interpretation of the data for the work.SWS and HTH: Substantial contributions to the conception and design of the work.TLZ: Revising this work critically for important intellectual content.MHW: Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.All authors read and approved the final manuscript. ",
"section_name": "Authors' contributions",
"section_num": null
},
{
"section_content": "Ethics approval and consent to participate Not available. ",
"section_name": "Declarations",
"section_num": null
},
{
"section_content": "Not available. ",
"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/s12883-018-1139-8
|
Targeting the Notch1 oncogene by miR-139-5p inhibits glioma metastasis and epithelial-mesenchymal transition (EMT)
|
Glioma metastasis, invasion, epithelial-mesenchymal transition (EMT) and chemoresistance indicate poor prognosis. Accumulating evidence reveals that Notch1 is an important factor in tumour progression. However, the role of Notch1 in glioma EMT and associated microRNAs (miRNAs) with the Notch pathway remain controversial.Utilizing cBioPortal database to examine the gene signature of NOTCH1 (encoding Notch1), CDH2 (encoding N-cadherin) and SNAI1 (encoding Snail-1) in disease-free survival (DFS) and overall survival (OS). We analyzed the Notch1 expression from Oncomine. We used Western blot (WB), immunohistochemistry (IHC) and immunofluorescence to determine protein levels. Transcription was evaluated by quantitative real-time (qRT)-PCR. siRNA and lentivirus were used to knock down Notch1 and overexpress miR-139-5p, respectively. The migration and invasion of glioma cells were assessed by wound healing and transwell assays. Luciferase reporter assays were utilized to verify the relationship between Notch1 and miR-139-5p. A U87-implanted intracranial model was used to study the effect of miR-139-5p on tumour growth and Notch1 suppression efficacy or EMT reversion.It revealed the association of NOTCH1, CDH2, SNAI1 genomic alterations with decreases in DFS and OS. Notch1 was upregulated in classical and proneural subtypes of GBM, and associated with tumour grade. Notch1 inhibition suppressed the biological behaviours of metastasis, invasion and EMT. Notch1 was identified as a novel direct target of miR-139-5p. MiR-139-5p overexpression partially phenocopied Notch1 siRNA, whereas the forced expression of Notch1 reversed the effects of miR-139-5p on the invasion of glioma. Moreover, intracranial tumourigenicity and EMT behaviours were reduced by the introduction of miR-139-5p and partially mediated by the decreased Notch1 expression.miR-139-5p was identified as a tumour suppressor by negatively targeting Notch1, and this work suggests a possible molecular mechanism of the miR-139/Notch1/EMT axis for glioma treatment.
|
[
{
"section_content": "Glioma is the most common primary malignant tumour of the central nervous system in adults [1,2].The high metastasis and invasiveness of glioma induce a high incidence of recurrence, which means a worse prognosis [3].Epithelial-mesenchymal transition (EMT) includes molecular changes, decreased cell-cell junction and adhesion, and increased cell motility.EMT can be determined by the loss of epithelial markers (E-cadherin) along with the upregulation of mesenchymal markers (N-cadherin, Fibronectin and Vimentin) [4]. Accumulating evidence shows that Notch1 plays an important role in tumour progress.The activation of Notch signalling by tenascin-C promotes the growth of human brain tumour-initiating cells [5].Notch1 activation is a poor prognostic factor in patients with gastric cancer [6].Recently, β-carotene has been reported to inhibit EMT though Notch pathway [7].NOTCH signaling is a primary inducer of EMT in a number of epithelial cancers, including cancer of the lung, breast and pancreas [8].D Maciaczyk et al. recently demonstrate that blocking Notch-pathway member CBF1 inhibits EMT-activator ZEB1 in glioma cells [9].However, little is known about the Notch1 interaction with EMT in glioma.Also, the molecular mechanisms remain elusive. MicroRNAs (miRNAs) are non-coding RNA molecules comprising 18~22 nucleotides [10].They regulate the expression of genes by directly targeting the 3′-untranslated regions (3′-UTR) of corresponding messenger RNAs (mRNAs) [11].miRNAs are involved in a variety of biological behaviours, including suppressing or promoting tumours.Among these, miR-139 inhibits the growth and metastasis of several cancers including myeloid leukaemia [12], laryngeal squamous carcinoma [13] and liver cancer through targeting, for example, c-Fos and CXCR4 [13,14].In particular, miR-139-5p suppresses cancer cell migration by targeting ZEB1 and ZEB2 in glioma [15].Our previous study confirmed that miR-139 was downregulated in clinical gliomas and glioma cell lines, and miR-139 inhibits Mcl-1 expression and potentiates TMZ-induced apoptosis in glioma [16].Few reports could be assessed until now, however, regarding the regulation of miR-139 on EMT in glioma, especially though Notch1. In this study, we attempted to investigate the expression and functions of Notch1 in gliomas and its relationship with miR-139-5p.For the first time, we showed that miR-139-5p reverses the Notch1-mediated EMT of glioma.This suggests an alternative for multiple treatments of glioma by regulating the miR-139-5p/Notch1/EMT pathway. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Twenty-nine human glioma tissues and four brain tissues were collected from patients who underwent surgical resection between January 2016 and March 2017 at the Second Affiliated Hospital of Harbin Medical University (HMU).Informed consent was obtained from all patients before the application of their tissue samples.This study complied with the regulations of Declaration of Helsinki and was approved by the medical ethics committee of HMU.All samples were graded histologically by clinical pathologists according to WHO guidelines, and they included 5 grade I tumours, 9 grade II tumours, 5 grade III tumours, and 10 grade IV tumours (Table 1).In addition, 4 normal adult brain tissue specimens were collected from patients who underwent severe traumatic brain injury and required surgical intervention (with informed consent). ",
"section_name": "Patients and specimens",
"section_num": null
},
{
"section_content": "LN229, U87, T98G and U251 glioma cell lines (human) were purchased from the Chinese Academy of Sciences cell bank.Oligodendroglia (Olig) was a gift from Fengmin Zhang, who is a professor of Harbin Medical University.These cells were cultured in a 5% CO 2 , 37 °C incubator in Dulbecco's Modified Eagle's Medium (DMEM, Corning, USA) supplemented with 10% foetal bovine serum (FBS, Biological Industries, Israel). ",
"section_name": "Cell culture",
"section_num": null
},
{
"section_content": "MiR-139-5p mimic was purified by high-performance liquid chromatography (GenePharma, Shanghai, China).Notch1 siRNA was composed and purchased from Invitrogen (USA) [17], and the sequences are listed in Additional file 1.The plasmid of full-length Notch1 without the corresponding 3′-UTR, pEGFP-N-Notch1 (GeneChem, Shanghai, China), was amplified and cloned into the GV230 (GeneChem, Shanghai, China).The plasmid along with miR-139-5p mimics or scramble were transfected into glioma cells with Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's instructions. Transfected cells were incubated for another 24-72 h at 37 °C with 5% CO 2 atmosphere.Afterwards, cells were harvested for RNA and protein analysis. ",
"section_name": "MicroRNAs, siRNA and plasmid transfection",
"section_num": null
},
{
"section_content": "To investigate the potential miRNAs that may regulate Notch1 mRNA, we utilized four commonly used miRNA databases, including miRanda algorithm (http://34.236.212.39/microrna/home.do),miRwalk (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/),Pictar (http://www.pictar.org/),and TargetScan (http:// www.targetscan.org/vert_71/). The TCGA data set within the cBioPortal database [18,19] (http://www.cbioportal.org/index.do) was extracted.The Glioblastoma Multiforme (GBM) cohort (TCGA, Provisional, n = 577), a merged cohort (TCGA, Cell 2016, n = 1084) of Brain Lower Grade Glioma (LGG) and GBM, the LGG cohort (TCGA, Provisional, n = 513) were utilized.NOTCH1 (encoding Notch1), CDH2 (encoding N-cadherin) and SNAI1 (encoding Snail-1) three-gene signature was then examined on independent cohorts above for effects on disease-free survival (DFS) and overall survival (OS). Notch1 mRNA expression from Oncomine (https:// www.oncomine.org/resource/login.html#) and the prognostic meaning of miR-139-5p in glioma from OncoLnc (http://www.oncolnc.org/)was extracted. ",
"section_name": "In silico analysis and establishing of a three-gene genomic signature",
"section_num": null
},
{
"section_content": "Total RNA was picked up using Trizol Reagent (Invitrogen, USA) according to the manufacturer's instructions.Total cDNA was reversely transcribed from 1 μg of total RNA (Perfect Real Time, Takara, Japan).Two-step qRT-PCR was performed for quantifying gene expression.We used a FastStart Universal SYBR Green Master (ROX) in the Roche LightCycler R Real-Time System.The expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6.The PCR conditions started at 95 °C for 15 s, then annealed and extended at 60 °C for 60 s.It is going on for 40 cycles followed by a melting curve analysis.The data was analysed by 2 -ΔΔCt method.All experiments were performed in triplicate.The primers used are shown in Additional file 2. ",
"section_name": "RNA isolation and quantitative real-time (qRT-PCR) assays",
"section_num": null
},
{
"section_content": "Cell lysates were harvested.Total protein of equivalent amounts were separated by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE).After that, they were transferred to polyvinylidene difluoride (PVDF) membranes.Block the membranes with 5% fat-free milk and 0.1% Tween-20 in tris-buffered saline with Tween (TBST) for 1.5 h.Next, the membranes were incubated with diluted anti-Notch1 (Abcam), E-cadherin (CST), N-cadherin (CST), Vimentin (Abcam), Fibronectin (Abcam), Snail-1 (Wanleibio), Shh (CST) and anti-GAPDH (Wanleibio) primary antibodies.Anti-rabbit or anti-mouse secondary antibodies (ZSGB-BIO), which were horseradish peroxidase-conjugated, were used and detected by the ECL system (Fujifilm Las-4000). ",
"section_name": "Western blotting assay",
"section_num": null
},
{
"section_content": "GV272-Notch1-3′-UTR (Genechem), a wild-type luciferase reporter plasmid was created.It contain a putative miR-139-5p binding sites as previously reported [20].Using Lipofectamine 2000 reagent (Invitrogen) to transfect these constructs into U87 or LN229 cells, with or without miR-139-5p mimics according to the manufacturer's protocol.miRNA mimics and firefly luciferase plasmid were co-transfected into cells.For normalization, they were co-transfected with CV045 Renilla luciferase plasmid (Genechem, Shanghai, China).Forty hours later, we used Dual-Glo luciferase assay system (E2920, Promega, USA) to measure the luciferase activity.The ratio of Firefly Luciferase activity to that of Renilla was defined as normalized luciferase activity. ",
"section_name": "Luciferase reporter assay",
"section_num": null
},
{
"section_content": "Cells were plated in 6-well plates.miR-139-5p or Notch1 siRNA were transfected into cells when confluency.A 200-μl sterile pipette tip was used to create scratches.Wash cells twice with PBS and then supply them with DMEM without FBS.Capture photographs at 0 h and after 24-36 h using an Axiovert 200 microscope (Carl Zeiss) and the data was analysed using Image pro-plus software. Transwell membranes was coated with Matrigel (BD Biosciences, San Jose, CA).About 5 × 10 4 cells/well were plated in the upper chamber.These cells were treated with miRNAs or Notch1 siRNA.The medium in upper chamber was serum-free.The medium in the lower chamber was 10% FBS.After 24 h, the cells in the top well was removed.The bottom cells were fixed with 95% ethanol, stained with 0.1% crystal violet.Take photographes in three independent 10× fields for each well.Three independent experiments were repeated. ",
"section_name": "Wound healing assay and transwell assay",
"section_num": null
},
{
"section_content": "Immunohistochemistry (IHC) and immunofluorescence assays were performed as previously described [21].IHC scores were assessed using a semiquantitative grading system [22].The appropriate antibodies against Notch1 (Abcam), E-cadherin (CST), N-cadherin (CST), Fibronectin (Abcam) and Vimentin (Abcam) were used.Immunofluorescence assays were visualized using Goat anti-Rabbit Alexa Fluor® 594-conjugated (ZSGB-BIO) or Goat anti-Mouse Fluorescein-conjugated (ZSGB-BIO) antibodies.Cell nuclei were counterstained using Hoechst 33258 (Thermo Fisher Scientific).Representative images were captured, and they were analysed by Olympus FV1000 Digital laser scanning microscopy. ",
"section_name": "Immunohistochemistry and immunofluorescence assay",
"section_num": null
},
{
"section_content": "U87 cells that were co-transducted with miR-139-5p lentivirus/scramble and luciferase lentivirus were injected intracranially into 5-week-old BALB/c-nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) as described earlier [23,24].Methods of animal care-taking and feeding were carried out according to the instructions of Beijing Vital River Laboratory Animal Technology (http://www.vitalriver.com/welfare.aspx).Exactly, they were cultured in SPF-class barrier system feeding conditions.The feed was disinfected with 121 degrees, 15 min, 1 kg pressure sterilization.Drinking water was filtered by multiple layers.Each group had 5 mice.After 20 days, the mice were sacrificed exposing to carbon dioxide.Continue to input carbon dioxide at a concentration of 100% for 2 min until the mouse stops breathing and then turns off the switch on the carbon dioxide bottle.Tumours were measured by fluorescent images of whole mice using an IVIS Lumina Imaging System (Xenogen).Portions of the tumour tissues were used to measure the Notch1 and EMT markers by IHC.Cryosections (4 mm) were used for IHC [1,22].These procedures were performed with approval by the Harbin Medical University Institutional Animal Care and Use Committee. ",
"section_name": "Xenograft assay",
"section_num": null
},
{
"section_content": "SPSS version 13.0 software (Chicago, IL, USA) was used to carried out all statistical analyses.Data were exhibited as means ± SD.Differences between the means of the treatment and control groups were analyzed using student's t-test.Significance among three or more groups was analyzed by a one-way analysis of variance (ANOVA).Categorical variables were compared using the χ 2 -test and Fisher's exact test.Data at p < 0.05 level were considered statistically significant.The survival curves were analysed using the log-rank test employing GraphPad Prism software. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "The three-gene signature correlated with decreases in DFS and OS in glioma We first explored the alteration frequency of NOTCH1 in different type of brain tumors (n = 1300) (cBioPortal) [18,19] and found the major type of genomic alterations in glioma was mutation or amplication (Fig. 1a andb).Then, we examined NOTCH1, CDH2 and SNAI1 three-gene signature in the Glioblastoma Multiforme (GBM) cohort (TCGA, Provisional, n = 577) (Fig. 1c) and demonstrated its association with decreases in DFS (P < 0.05) (Fig. 1d).This association was also revealed in a merged cohort (TCGA, Cell 2016, n = 1084) of Brain Lower Grade Glioma (LGG) and GBM (P < 0.001) (Fig. 1e andf ).However, the association was not significant in the LGG cohort (TCGA, Provisional, n = 513) (p = 0.588) (Fig. 1g). ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "We first extracted the data from Oncomine.From Bredel Brain and Sun Brain data sets, we analyzed Notch1 mRNA expression.The result indicated that GBM samples overexpressed more Notch1 (Fig. 2a).Further, we analyzed the mRNA microarray data from TCGA.It suggested that Notch1 were significantly upregulated in classical and proneural subtypes of GBM (Fig. 2b).Notch1 expression was measured using an immunohistochemical analysis in 29 different grades of glioma tissues and 4 normal brain tissues.In Grade III-IV tissues, Notch1 was higher than that in low grade gliomas (WHO II) or normal brain tissues (P < 0.05) (Fig. 2c).Furthermore, the expression patterns of Notch1 were confirmed by Western blotting assay in 4 glioma cell lines (LN229, U87, T98G and U251).Glioma cells, especially U87 and LN229 cells, expressed more Notch1 compared with Olig (Fig. 2d).In addition, we assessed the correlation between Notch1 expression and clinicopathologic characteristics in 33 patients and found that Notch1 expression was positively correlated with tumour grade and negatively correlated with Karnofsky Performance status (KPS) score (P < 0.05, Table 2). ",
"section_name": "Notch1 was upregulated in glioma tissues and cell lines and associated with tumour grade",
"section_num": null
},
{
"section_content": "Given that Notch1 is highly expressed in glioma and a crucial regulator of epithelial-mesenchymal-transition (EMT), we subsequently investigated its biological importance on the tumourigenic property of glioma cells, including metastasis and invasion.We knocked down Notch1 in LN229 and U87 cells (Fig. 3a and b, Fig. 6c andd) and then performed a wound-healing assay and transwell assay to test invasive characteristics.The results showed that siNotch1 attenuated cell migration (Fig. 3c and e for LN229, Fig. 3d and f for U87) and decreased the number of invasive glioma cells compared with the scramble siRNAs (Fig. 3g and i for LN229, Fig. 3h and j for U87). ",
"section_name": "Notch1 knockdown suppressed metastasis and invasion capability of glioma cells",
"section_num": null
},
{
"section_content": "To investigate whether microRNAs were involved in regulating Notch1, we used the Targetscan, miRanda, Pictar and miRwalk databases and identified potential miR-NAs, including miR-139-5p (Fig. 4a), that target Notch1 3′-UTR.Accordingly, we transfected miR-139-5p mimics into glioma cells and evaluated the Notch1 expression level.Transfection efficiency was evaluated using qRT-PCR (Additional file 3).qRT-PCR and Western blotting showed that miR-139-5p induced an obvious decline in Notch1 expression (Fig. 4b andc).Further, we sought to confirm whether Notch1 was a direct target of miR-139-5p.GV272-Notch1-3′-UTR, luciferase reporter plasmid was constructed.It contained a putative miR-139-5p binding site (Fig. 4d).We transfected these plasmids into glioma cells with miRNAs.The data showed that luciferase activity decreased in the group of WT-Notch1-3′-UTR and miR-139-5p mimics.No significant change in any other group (Fig. 4e andf).These data suggest that miR-139-5p binds to the 3′-UTR of Notch1 directly. ",
"section_name": "Notch1 was a direct target of miR-139-5p",
"section_num": null
},
{
"section_content": "After confirming the relationship between Notch1 and miR-139-5p, we intended to test the effect of miR-139-5p on invasive activity.The wound-healing assay and transwell assay showed that miR-139-5p attenuated cell migration (Fig. 5a andb) and decreased the number of invasive glioma cells compared with the scramble miRNAs (Fig. 5c andd).Moreover, miR-139-5p downregulated the mesenchymal markers (N-cadherin, vimentin and fibronectin) but upregulated the epithelial marker (E-cadherin) at both the mRNA and protein levels (Fig. 5e, f, g, h and i), which indicated an EMT-suppressive role of miR-139-5p. ",
"section_name": "Overexpressed miR-139-5p inhibited glioma metastasis, invasion and EMT",
"section_num": null
},
{
"section_content": "To further investigate the mechanism of miR-139-5p on glioma suppression, we sought to determine whether the anti-EMT effects of miR-139-5p are mediated by Notch1.To address this, we treated U87 and LN229 with Notch1 siRNA followed by a rescue experiment. The qRT-PCR and Western blotting assay confirmed specific knockdown of Notch1 by siRNA (Fig. 6a-d). Notch1 siRNA dramatically decreased the mesenchymal markers (N-cadherin, vimentin and fibronectin) while increasing the epithelial marker E-cadherin (Fig. 6a-d). Snail-1 is a zinc finger transcription factor that can repress E-cadherin transcription [25,26].Sonic hedgehog (Shh) is one of the stem cell-associated protein [27].The results also showed Notch1 siRNA significantly decreased the expression of Snail-1 and Shh (Fig. 6c andd).In addition, in the treatment with full-length Notch1 without the corresponding 3′-UTR and followed by miR-139-5p mimics for 48 h, we found that miR-139-5p partially inhibits forced Notch1 expression in glioma cells (Fig. 6e andf).Furthermore, the effects of Notch1 on EMT markers after overexpression of miR-139-5p were also examined.The result showed forced expression of Notch1 reversed the effects of miR-139-5p on EMT markers (Fig. 6e andf ).Accordingly, the upregulation of Notch1 significantly rescued the glioma invasion behaviour (Fig. 6g andh).Taken together, these data indicated that Notch1 was a mediator of the EMT-suppressive role of miR-139-5p. ",
"section_name": "Mir-139-5p reversed EMT via down-regulating the expression of Notch1",
"section_num": null
},
{
"section_content": "To evaluate the antiglioma effect of miR-139-5p in vivo, a U87 xenograft model was used.We found that miR-139-5p-treated cells significantly reduced tumour size (P < 0.05, Fig. 7a andb).The Kaplan-Meier curve analysis showed a marked longer survival period of the miR-139-5p-treated group compared with the scramble group (P < 0.05, Fig. 7c).In the meantime, miR-139-5p decreased the expression of Notch1 as well as mesenchymal markers (N-cadherin, vimentin, fibronectin) while increasing E-cadherin (Fig. 7d). ",
"section_name": "MiR-139-5p inhibited glioma xenograft growth, metastasis and EMT in vivo and prolonged survival",
"section_num": null
},
{
"section_content": "Glioblastoma is characterized by a high capacity to proliferate and invade.Gliomas that metastasize often have poor prognosis [28,29].The search for effective drugs that can suppress glioma metastasis has been a main topic of clinician research.The Notch signalling pathway plays an important role in cell fate determination during normal development [30].Notch1 has tumour-suppressing and promoting functions in human prostate cancer [31] or in different tumours [5,32].A combination of Notch1 blockade and chemotherapy synergistically reduced chemotherapy-enriched cancer stem cells (CSC) [33].Blocking Notch-1 resulted in downregulation of NF-kappaB and its target genes (CXCL8, MMP9 and VEGF), which suppressed invasion and angiogenesis in breast cancer [34]; therefore, we would evaluate these targets next in glioma cell lines and in any other experiments where miR-139-5p levels are manipulated.Sonic hedgehog (Shh) could promote tumour proliferation in a Notch-dependent manner [35][36][37][38].The Hedgehog and Notch pathways interact to control the EMT/MET [35].However, little is known regarding Notch1 interactions with EMT in glioma.Our study demonstrated that Notch1 was obviously upregulated in glioma tissues.Interestingly, Notch1 also expressed in normal brain tissue, which could be explained by Notch1 signalling being involved in cell fate decision during normal development while abnormal activation would promote carcinogenesis [30].In addition, Notch1 was upregulated in glioma cell lines, especially U87 and LN229.Knocking down Notch1 in these cells effectively suppressed glioma metastasis, invasion and EMT.These results demonstrated that Notch1 plays an important role in glioma and could be a potential therapeutic target.MiR-139-5p has been demonstrated as a tumour suppressor in a variety of tumours.Krowiorz et al. found that miR-139-5p is specifically downregulated in CN-AML with mutated FLT3 and acts as a strong tumour suppressor [12].Wang et al. reported that miR-139 functions as an anti-oncomir to repress glioma progression through targeting IGF-1R, AMY-1, and PGC-1beta [39].Moreover, miR-139-5p can sensitize colorectal cancer cells to 5-fluororacil by targeting NOTCH-1 [40].Until now, limited information is available about the effect of miR-139-5p on EMT in glioma.miR-139-5p had prognostic meaning in LGG (Additional file 4).Our recent work demonstrates that miR-139 is downregulated in glioma tissues and negatively correlated to tumour grade [16].In this study, we searched four databases to find that miR-139-5p may target Notch1 3ˊ-UTR.The combination of bioinformatics prediction, luciferase reporter assays and functional experiments determined that miR-139-5p decreased Notch1 expression degradation, low efficiency in crossing the blood-brain barrier, side effects and the off-targeting of miR-139-5p. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Notch1 is markedly overexpressed in glioma and accelerated tumour metastasis, invasion and EMT.Upregulating miR-139-5p in cells inhibits glioma growth and reverses Notch1-induced EMT.This suggests that the miR-139-5p/Notch1/EMT pathway could be a novel target for glioma therapy. ",
"section_name": "Conclusion",
"section_num": null
}
] |
[
{
"section_content": "We thank Professor Quan Liu and Professor Jinquan Cai (HMU) for guidance on the study. This study was approved by the Ethics Committee of the Second Affiliated Hospital of Harbin Medical University (HMU).The review coding is 2013-R-024.Informed consent was written from all patients before the application of their tissue samples.This study conformed to the standards set by the Declaration of Helsinki and was approved by the medical ethics committee of HMU.The study participants agreed to participate and provided written informed consent.These animal experiment procedures were performed with approval by the Harbin Medical University Institutional Animal Care and Use Committee. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "We thank Professor Quan Liu and Professor Jinquan Cai (HMU) for guidance on the study. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This study was approved by the Ethics Committee of the Second Affiliated Hospital of Harbin Medical University (HMU).The review coding is 2013-R-024.Informed consent was written from all patients before the application of their tissue samples.This study conformed to the standards set by the Declaration of Helsinki and was approved by the medical ethics committee of HMU.The study participants agreed to participate and provided written informed consent.These animal experiment procedures were performed with approval by the Harbin Medical University Institutional Animal Care and Use Committee. ",
"section_name": "Ethical approval and consent to participate",
"section_num": null
},
{
"section_content": "This work was supported by the National Natural Science Foundation of China (81572743), President Foundation of Nanfang Hospital, Southern Medical University (2017B030), National Natural Scientific Fund (81372173, 81502404), the Scientific Fund Project of Hospital (KYBS2015-15) and the HLJ Province Natural Scientific Fund (QC2015128). ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "Data are stored by the corresponding author of this paper and are available upon request.Databases included miRanda algorithm (http://34.236.212.39/microrna/home.do),miRwalk (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/), Pictar (http://www.pictar.org/),TargetScan (http:// www.targetscan.org/vert_71/),TCGA (https://cancergenome.nih.gov/),cBioPortal (http://www.cbioportal.org/index.do),Oncomine (https:// www.oncomine.org/resource/login.html#) and OncoLnc (http:// www.oncolnc.org/). ",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "Authors' contributions LR, JC and CL conceived and designed this study.LJ, LQ and LL drafted the manuscript and performed the statistical analysis.WR and DW participated in the clinical evaluation of the patients.All authors approved this manuscript to be submitted. 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": "Authors' contributions LR, JC and CL conceived and designed this study.LJ, LQ and LL drafted the manuscript and performed the statistical analysis.WR and DW participated in the clinical evaluation of the patients.All authors approved this manuscript to be submitted. ",
"section_name": "Additional files",
"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
}
] |
10.1038/s41375-019-0557-y
|
BCR signaling contributes to autophagy regulation in chronic lymphocytic leukemia
|
No abstract available.
|
[
{
"section_content": "Macroautophagy (herein Autophagy) maintains cellular homeostasis through bulk protein degradation.During the autophagy process LC3 (microtubule associated protein 1 light chain 3) and GABARAP (GABA type A receptor proteins) family proteins, ATG3 and ATG7 are essential for autophagosome formation, autophagosome-lysosome fusion, and cargo degradation (Supplementary Fig. S1) [4].In CLL, high basal expression of autophagy genes, BECN1, PIK3C3, and PIK3R4, have been associated with a shorter time-to-first treatment (TTFT) and worse overall survival in patients [5].Furthermore, BECN1 and ATG5 RNA expression have been shown to correlate with poorer clinical outcome [6] and autophagy was shown to reduce therapyinduced apoptosis [7].However, how BCR activation regulates autophagy in CLL has not been previously evaluated. Evaluation of basal autophagy-protein levels demonstrated significantly greater LC3B-II, GABARAPL2, ATG3, and ATG7 in CLL cases (Supplementary Table S1; for antibody information, see Supplementary Table S2) compared with healthy donor B-cells (HDB) (Fig. 1a and Supplementary Fig. S2A).The basal levels of LC3B-II, but not GABARAPL2, ATG3, or ATG7, were significantly greater still in U-CLL compared with M-CLL (Fig. 1b and Supplementary Fig. S2B).In addition, LC3B-II and ATG3 levels, but not GABARAPL2 or ATG7, associated with BCR signaling capacity (Fig. 1c and Supplementary Fig. S2C).With significantly greater levels of LC3B-II and ATG3 observed within U-CLL compared with MCLL-LS due to the contrasting extremes of anti-IgM-mediated signaling between these two subgroups.M-CLL-S samples that retained anti-IgM signaling capacity expressed LC3B-II at an intermediate level between U-CLL and M-CLL-LS cases (Fig. 1c), suggesting LC3B-II levels were influenced by both inherent BCR signaling and the cells' origin.Indeed, greater LC3B-II levels were associated with a significantly shorter TTFT from the first lymphocytosis (Supplementary Fig. S3A) irrespective of IGHV mutational status, as both cohorts expressed a mixture of U-CLL and M-CLL cases.Intriguingly, although numbers were small, LC3B-II expression also appeared to identify M-CLL patients, but not U-CLL patients, which required earlier treatment (Supplementary Fig. S3B).However, further work in a larger trial using a fully quantitative technique is required to identify the potential of LC3B-II as a biomarker. To determine whether autophagy occurred in vivo, we performed immunohistochemistry on lymph node tissue from CLL patients and control tissue from healthy, reactive nodes.LC3B expression was detected most strongly within proliferation centers from sequential tissue sections labeled with Ki67 (Supplementary Fig. S4A,B) and in reactive node germinal centers (Supplementary Fig. S4D).This shows that LC3B expression is associated with CLL cells and B-cells that had undergone antigen engagement.These cells appeared to be B-cells given that the majority of cells were CD20+ in sequential tissue sections (Supplementary Fig. S4C,D). Consequently, we assessed whether BCR engagement for 2, 4, or 24 h with bead-bound anti-IgM or anti-IgD could modify expression of key autophagy proteins LC3B-II, GABARAPL2, ATG3, and ATG7 in CLL cells (Fig. 1d and Supplementary Fig. S5A,B).Significant increases in the level of all proteins were observed 24 h following treatment with anti-IgM.However, significant increases in GABAR-APL2 and LC3B-II also occurred earlier between 2 and 4 h, whereas ATG3 and ATG7 only demonstrated trends for increased expression at this time.Subsequent evaluation of LC3B-II expression in a larger cohort of patients treated with bead-bound anti-IgM for 24 h demonstrated that the majority of CLL cases activated their autophagy pathway in response to BCR engagement (Supplementary Fig. S5C).Furthermore, U-CLL cases expressed greater levels of LC3B-II in response to anti-IgM compared with M-CLL cases (median fold increase of 5.4 and 3.5, respectively; Fig. 1e).Some overlap in the anti-IgM-mediated LC3B-II levels was identified between IGHV subsets that maybe a result of M-CLL cases with active BCR signaling.Compared with anti-IgM, anti-IgD-induced signaling is short-lived in CLL cases [8].Therefore, although significant increases in LC3B-II were observed following bead-bound anti-IgD treatment at all time points, with trends for increased GABARAPL2, in most instances these increases were smaller than that observed with anti-IgM (Supplementary Fig. S5D).Similarly, a small but significant increase in ATG7 levels were observed at 24 h with bead-bound anti-IgD, with only a trend for increased ATG3 expression at 24 h (Supplementary Fig. S5D). We subsequently characterized the signaling pathways involved in BCR-mediated autophagy (Supplementary Fig. S6).Phospho-ERK and -AKT levels increased 15 min post stimulation with bead-bound anti-IgM or anti-IgD, which is indicative of active BCR signaling.Interestingly, anti-IgM and anti-IgD increased phospho-p70 S6 kinase levels at 6 h, indicating active mammalian target of rapamycin signaling, despite an induction of LC3B-II expression, suggesting activation of non-canonical autophagy [9].At 24 h, anti-IgM-and anti-IgD-dependent increases in LC3B-II, p62, and phospho-ATG13 suggested simultaneous activation of canonical and non-canonical autophagy [9,10]. Next, we assessed autophagosome formation using LC3B puncta formation by immunofluorescence [11].Basal autophagy was observed in cells treated with bead-bound control antibody (Supplementary Fig. S7A,B), whereas treatment with bead-bound anti-IgM increased LC3B puncta to levels above that seen in the control (Supplementary Fig. S7A,B). To confirm that bead-bound anti-IgM-mediated autophagy was BCR-mediated and not a bead-dependent affect, CLL cells were treated with bead-bound or soluble anti-IgM in the presence or absence of the autophagosome inhibitor hydroxychloroquine (HCQ).Both soluble and bead-bound anti-IgM significantly increased LC3B-II and GABAR-APL2 levels compared with the control (Supplementary Fig. S8A,B), confirming that the induction of autophagy proteins was mediated by BCR engagement and was not a bead-dependent effect.Treatment with soluble anti-IgM resulted in smaller autophagy protein increases in the same CLL samples compared with bead-bound anti-IgM, likely due to the relatively higher BCR signal strength and duration with bead-bound verse soluble anti-IgM [12]. To determine whether the anti-Ig-dependent regulation of autophagy was B-cell or tumor specific, we treated HDB with bead-bound or soluble anti-IgM or anti-IgD for 24 h.Bead-bound anti-IgM, and to a lesser extent anti-IgD, promoted significant increases in LC3B-II levels (Supplementary Fig. S9A,B), suggesting that this phenomenon was Bcell and not tumor-specific.However, in contrast to CLL, these changes were not consistently replicated in the other autophagy markers examined. To determine whether BCR engagement blocked LC3B-II degradation or induced autophagosome formation, we assessed autophagic flux in response to bead-bound anti-IgM using immunoblotting in the presence or absence of HCQ as described in the autophagy guidelines [11].CLL cells treated with HCQ accumulated LC3B-II in a concentration-dependent manner, which was further augmented with bead-bound anti-IgM (Fig. 2a).The LC3B-II increases were not a result of HCQ-mediated effects on BCR signaling (Supplementary Fig. S10) and also occurred in a time-dependent manner at the RNA level (Supplementary Fig. S11A).Comparatively, bead-bound anti-IgD stimulation produced similar but much smaller responses than anti-IgM (Supplementary Fig. S11B).These data confirm that BCR engagement does not block autosomal degradation but induces the expression of autophagyassociated genes leading to increased autophagic flux. Next, we used interleukin (IL)-4 and BCR kinase inhibitors to confirm the role of BCR signaling in the regulation of BCR-mediated autophagy in CLL.We previously demonstrated that IL-4 induced surface IgM (sIgM) expression and subsequent downstream signaling [13].IL-4 treatment significantly increased sIgM expression as previously demonstrated and augmented anti-IgM-dependent LC3B-II levels (Fig. 2b and Supplementary Fig. S12A,B).These affects appeared to be BCR-mediated, as IL-4 had no substantive effect on LC3B-II levels alone, even in the presence of HCQ.Next, we inhibited BCR signaling with tamatinib (SYK) and Ibrutinib (BTK), and observed the effect on LC3B-II levels.Both inhibitors significantly reduced LC3B-II to basal levels (Fig. 2c and Supplementary Fig. S12C).These data confirm the role of BCR signaling in the regulation of autophagy in CLL. Previous studies have shown a role for autophagy in resistance to venetoclax-mediated killing in follicular lymphoma [14].Therefore, we hypothesized that autophagy inhibitors may synergize with established therapies to inhibit basal, BCR-induced, or therapy-induced autophagy in CLL.To address this, we examined the effect on CLL cell viability of combining the autophagy inhibitor, VPS34-IN1, with venetoclax following bead-bound anti-IgM treatment.We observed significant synergy between VPS34-IN1 and venetoclax compared with single-agent treatment (Fig. 2d,e).Importantly, VPS34-IN1 inhibited BCR-mediated LC3B-II increases but had no effect on BCR signaling (Supplementary Fig. S13A,B), and both ABT-199 and VPS34-IN1-mediated cell death was largely caspase-dependent (Fig. 2d).These data indicate a protective cellular effect of BCR-mediated autophagy in CLL and highlight the therapeutic potential of inhibiting autophagy pathways to promote greater CLL cell killing. 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/.Fig. 2 BCR-mediated autophagy is dependent on BCR signaling and provides a survival advantage to CLL cells.a CLL samples (n = 11) were treated with bead-bound isotype control antibody (IC) or anti-IgM with or without HCQ at indicated concentrations for 24 h and LC3B-II levels evaluated by immunoblotting.A representative immunoblot is shown.Blots were quantified and the mean fold change (±SEM) in LC3B-II level with each treatment vs. IC without HCQ is shown in the accompanying graph.A t-test was used for statistical analysis.b CLL samples (n = 6) were treated with or without IL-4 (10 ng/ml), in the presence or absence of HCQ, for 24 h before treatment with bead-bound IC or anti-IgM for 24 h.LC3B-II levels were evaluated by immunoblotting and the mean fold change (±SEM) in LC3B-II levels with each treatment vs. IC without HCQ is shown.A Wilcoxon's matched-pairs signed-rank test was used for statistical analysis.c CLL samples (n = 10) were treated with HCQ and a SYK (tamatinib; Tam) or BTK (ibrutinib; Ibr) inhibitor (both 5 μM) for 1 h before stimulation with bead-bound IC or anti-IgM for 24 h and the LC3B-II level evaluated by immunoblotting.Hsc70 was used as a loading control.A representative immunoblot is shown.Blots were quantified and the mean fold change (±SEM) in LC3B-II level with each treatment vs. IC DMSO is shown in the accompanying graph.A Wilcoxon's matched-pairs signed-rank test was used for statistical analysis.d CLL samples were treated for 6 h with bead-bound IC or anti-IgM before treatment with autophagy inhibitor, VPS34-IN1 (3 μM), either alone or in combination with venetoclax (5 nM n = 6, or 10 nM n = 8 where shown) for 24 h.All conditions were carried out with or without Q-VD-Ph (10 μM) to identify caspase-dependent drugmediated cell killing.Cell viability was assessed by the CellTiter-Glo Cell Viability Assay.The mean (+SEM) percentage of viable cells relative to IC is shown.A Wilcoxon's matched-pairs signed-rank test was used for statistical analysis.e Synergy between VPS34-IN1 (3 μM) and venetoclax (5 nm, left and 10 nM, right) was evaluated as detailed in the Supplementary Materials and Methods.XY line, observed survival = expected survival.Points below the line, synergistic interactions; points above the line, additive interactions ",
"section_name": "",
"section_num": ""
}
] |
[
{
"section_content": "Acknowledgements We thank Bloodwise (12044, 14040, 16003), CRUK (C2750/A23669), the patients for supplying tissue, the support from CRUK center grant (C34999/A18087), and ECMC grant (C24563/A15581).We thank Dr Ian Tracy and Dr Kathy Potter, Mrs Isla Henderson, and Ms Carina Mundy for sample characterization and storage.We thank Dr Sonya James and the University of Southampton Biomedical Imaging Unit.We thank Professor Sharon Tooze for reading the manuscript and providing scientific guidance.Finally, we acknowledge Professor Tessa Holyoake who was a collaborator on this study but who passed away before project completion. ",
"section_name": "Compliance with ethical standards",
"section_num": null
},
{
"section_content": "Acknowledgements We thank Bloodwise (12044, 14040, 16003), CRUK (C2750/A23669), the patients for supplying tissue, the support from CRUK center grant (C34999/A18087), and ECMC grant (C24563/A15581).We thank Dr Ian Tracy and Dr Kathy Potter, Mrs Isla Henderson, and Ms Carina Mundy for sample characterization and storage.We thank Dr Sonya James and the University of Southampton Biomedical Imaging Unit.We thank Professor Sharon Tooze for reading the manuscript and providing scientific guidance.Finally, we acknowledge Professor Tessa Holyoake who was a collaborator on this study but who passed away before project completion. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Compliance with ethical standards",
"section_num": null
},
{
"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. ",
"section_name": "Conflict of interest",
"section_num": null
}
] |
10.1186/1749-8104-6-35
|
Basal progenitor cells in the embryonic mouse thalamus - their molecular characterization and the role of neurogenins and Pax6
|
<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>The size and cell number of each brain region are influenced by the organization and behavior of neural progenitor cells during embryonic development. Recent studies on developing neocortex have revealed the presence of neural progenitor cells that divide away from the ventricular surface and undergo symmetric divisions to generate either two neurons or two progenitor cells. These 'basal' progenitor cells form the subventricular zone and are responsible for generating the majority of neocortical neurons. However, not much has been studied on similar types of progenitor cells in other brain regions.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>We have identified and characterized basal progenitor cells in the embryonic mouse thalamus. The progenitor domain that generates all of the cortex-projecting thalamic nuclei contained a remarkably high proportion of basally dividing cells. Fewer basal progenitor cells were found in other progenitor domains that generate non-cortex projecting nuclei. By using intracellular domain of Notch1 (NICD) as a marker for radial glial cells, we found that basally dividing cells extended outside the lateral limit of radial glial cells, indicating that, similar to the neocortex and ventral telencephalon, the thalamus has a distinct subventricular zone. Neocortical and thalamic basal progenitor cells shared expression of some molecular markers, including <jats:italic>Insm1</jats:italic>, Neurog1, Neurog2 and NeuroD1. Additionally, basal progenitor cells in each region also expressed exclusive markers, such as Tbr2 in the neocortex and Olig2 and Olig3 in the thalamus. In <jats:italic>Neurog1</jats:italic>/<jats:italic>Neurog2</jats:italic> double mutant mice, the number of basally dividing progenitor cells in the thalamus was significantly reduced, which demonstrates the roles of neurogenins in the generation and/or maintenance of basal progenitor cells. In <jats:italic>Pax6</jats:italic> mutant mice, the part of the thalamus that showed reduced Neurog1/2 expression also had reduced basal mitosis.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p>Our current study establishes the existence of a unique and significant population of basal progenitor cells in the thalamus and their dependence on neurogenins and Pax6. These progenitor cells may have important roles in enhancing the generation of neurons within the thalamus and may also be critical for generating neuronal diversity in this complex brain region.</jats:p> </jats:sec>
|
[
{
"section_content": "The immense number of neurons in the mammalian neocortex is thought to be determined during development by a prominent progenitor cell population that shows a distinct pattern of migration and division.Unlike the predominant progenitor cell type in other brain regions, the radial glial cells (RGs), these cells divide basally away from the ventricular surface and undergo a symmetric division that generates two neurons or two progenitor cells.It is thought that the six-layered mammalian neocortex is largely dependent on division of these basal progenitor cells that serve as transit amplifying cells or intermediate progenitor cells (IPCs), and that the evolution of the mammalian cortex is correlated with the emergence of progenitor cell populations that enhance the generation of neurons [1][2][3][4]. Basally dividing progenitor cells have been identified not only in the cerebral cortex, but also in ganglionic eminences, thalamus, hindbrain and spinal cord [5][6][7][8][9].However, only the cerebral cortex and ganglionic eminences have been shown to harbor a robust enough population of basal progenitor cells to form a distinct domain, the subventricular zone (SVZ), above the domain of RGs that comprises the ventricular zone (VZ).Recent studies identified a number of molecular markers of basal progenitor cells in the developing neocortex.In addition, genes such as Tbr2 [10,11], Insm1 [12] and AP2g [13] or inhibition of Notch signaling [14,15] are found to be essential for the generation of basal progenitor cells from RGs. Time-lapse analysis of fluorescently labeled cortical progenitors in slices elucidated the unique migratory patterns and modes of division of neocortical basal progenitor cells and showed that these cells function as transit amplifying progenitor cells, or IPCs [9,16,17]. The mammalian thalamus has an extremely complex organization with several dozen distinct neuronal populations called nuclei [18].During embryogenesis, the thalamus is composed of two molecularly distinct domains of neural progenitor cells, pTH-C and pTH-R, located across rostro-caudal and dorso-ventral axes [19].pTH-C is a larger, caudo-dorsally located domain that expresses the basic helix-loop-helix (bHLH) transcription factors neurogenin 1 (Neurog1) and neurogenin 2 (Neurog2) and gives rise to all of the thalamic nuclei that project to the cortex.pTH-R is a smaller domain that expresses another bHLH protein, Ascl1 (also known as Mash1), and lies between pTH-C and the zona limitans intrathalamica (ZLI), the boundary population that abuts the thalamus and the prethalamus [19].pTH-R likely contributes to the majority of GABAergic neurons in the thalamus, including part of the ventral lateral geniculate nucleus and intergeniculate leaflet.Recent studies have unveiled critical roles of secreted signaling molecules in the formation of positional diversity of thalamic progenitor cells [20][21][22][23][24]. Despite the finding that there are basally dividing cells in embryonic mouse thalamus [6], their molecular characteristics and the mechanisms for their generation have not yet been determined.Considering the extensive connections between the thalamus and neocortex, we anticipated that the mammalian thalamus has diversified its progenitor cell populations during evolution to allow generation of a larger number of neurons comparable to those found in the six-layered neocortex. In the study described here, we explored this possibility by performing a detailed characterization of thalamic basal progenitor cells in mouse embryos.We found that the thalamus contains a remarkably large number of basal progenitor cells, some of which form the SVZ similar to that found in the neocortex and ventral telencephalon.Thalamic basal progenitor cells do not express the same molecular markers as neocortical IPCs, such as Tbr2, but they do share many other aspects with their putative cortical counterpart, including the expression of the bHLH transcription factors NeuroD1 and neurogenins (Neurog1 and Neurog2).We also show the first evidence that Neurog1 and Neurog2 are required for the normal number of basally dividing cells, which demonstrates the critical role of these transcription factors in the formation and/or the maintenance of thalamic basal progenitor cells. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "The embryonic mouse thalamus contains a large number of basally dividing cells We found numerous cells away from the surface of the third ventricle that express the M-phase marker phosphorylated histone H3 (PH3), which we define as dividing basal progenitor cells (Figure 1A-E, arrowheads).These cells were found as early as at embryonic day (E)10.5 (Figure 1A,B) and persisted until at least E14.5 (Figure 1E).Double/triple immunohistochemistry showed that most of these basal progenitor cells are within the progenitor domain pTH-C, which gives rise to all of the thalamic nuclei that project to the cerebral cortex [19] (Figure 1B, marked as 'C').Within the pTH-C domain, the ratio of basal PH3-positive cells to total PH3-positive cells was highest at E12.5 and declined at E14.5, when thalamic neurogenesis is largely complete, except in the most dorsal location (Figure 1E-G) [25].In contrast, fewer PH3-positive cells were found in the progenitor domain pTH-R, which produces neurons that do not project to the cortex [19] (Figure 1B, marked as 'R') and in the ZLI, the border cell population abutting the thalamus and the prethalamus (Figure 1B, marked as 'ZLI').The ratio of PH3-positive cells in the basal location to the total PH3-positive cells was significantly higher in pTH-C than the other two domains analyzed (pTH-R and ZLI; Figure 1H).Figure 1I shows the average number of PH3-positive cells in each of the 20 μm-wide medial-lateral bins within pTH-C at E12.5.In addition to the high peak at the ventricular (apical) surface (bin 1), there was another peak of PH3-expressing cells away from the third ventricle (bin 6), indicating the presence of a discrete population of thalamic progenitor cells (Figure 1I).These initial analyses demonstrate the presence of basally dividing progenitor cells in the thalamus throughout neurogenesis and that they are particularly enriched in the progenitor domain pTH-C. ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "We next asked if the basally dividing cells comprise a distinct zone in the mouse thalamus that is not populated by RGs.Such a zone, the SVZ, emerges in mice by E14.5 in the neocortex, and by E13.5 in the ganglionic eminences [26]; however, it has not been evaluated in other brain regions.We used NICD (intracellular domain of Notch) and Pax6 as markers of RGs.NICD is a cleavage product of Notch1 [27], which is co-expressed with Nestin within the neocortical VZ, but not in the SVZ [28].Notch activity inhibits the formation of IPCs from RGs in the neocortex, indicating that the presence of NICD is a marker for RGs within the VZ.Pax6 is also highly expressed in neocortical RGs in the VZ [29], although low-levels of Pax6 expression are detectable in many IPCs [29] and a recent report identified a new class of Pax6-expressing progenitor cells that divide away from the lateral ventricle in the mouse neocortex [30]. We found that NICD is expressed in a cluster of cells near the third ventricle at both E11.5 (Figure 2A,C,C', left of dashed line) and E12.5 (Figure 2E,G,G', left of dashed line).Basal PH3-positive cells were located on both sides of the lateral margin of this NICD cluster at E11.5 and E12.5 (Figure 2A,E; arrows indicate the outside population and arrowheads indicate the inside population).The outer population became more evident at E12.5 (Figure 2E).PH3-positive cells were also observed on both sides of the Pax6 domain (Figure 2B,F, arrowheads and arrows).Similar to the neocortex, more laterally located basal progenitor cells expressed low levels of Pax6 (Figure 2B,D,D' at E11.5; 2F,H,H' at E12.5).In addition, Pax6 expression was generally lower in the rostral part of the pTH-C domain at both E11.5 and E12.5 (Figure 2B,F, bracket).Labeling of S-phase cells with a 0.5-hour ethynyl deoxyuridine (EdU) pulse showed that some thalamic progenitor cells reside outside the NICD + /Pax6-high zone (Figure 2C',D'G'H').Based on these results, we propose that, as early as E11.5, a molecularly distinguishable SVZ exists in the pTH-C domain of the thalamus, which we define as the zone where progenitor cells exist outside of the NICD + /Pax6-high VZ.Thalamic basal progenitor cells populate both the VZ and SVZ. ",
"section_name": "The embryonic mouse thalamus has a defined subventricular zone",
"section_num": null
},
{
"section_content": "We then examined the expression patterns of previously characterized genes that are expressed in thalamic progenitor cells in order to determine the progenitor zone (VZ or SVZ) and progenitor cell types (RGs or basal progenitor cells) in which each gene is expressed (Figures 3, 4 and5).Thalamic progenitor cells ubiquitously express the bHLH transcription factor Olig3 [19], but the neocortex does not.Double staining with a 0.5-hour EdU pulse showed that the domain of Olig3 expression in the thalamus encompassed the entire medial-lateral extent of thalamic progenitor cells, indicating that Olig3 is expressed in both the VZ and SVZ of the thalamus (Figure 3A,F).In addition, we found that Olig3 heavily overlaps with NICD (Figure 5A), demonstrating that Olig3 is expressed in RGs.Together, these results show that Olig3 is expressed in both the VZ and SVZ and in both RGs and basal progenitor cells in the thalamus. We next determined if NeuroD1 and Insm1 (insulinoma-associated 1), markers for neocortical IPCs, are also expressed in the thalamus.NeuroD1 is a bHLH transcription factor that is expressed in the upper SVZ and lower intermediate zone of the neocortex, presumably being induced following Tbr2 expression [31].In the pTH-C domain of the thalamus, a densely packed population of NeuroD1-positive cells was found in the middle portion of the diencephalic wall (Figure 3B, arrow).In addition, some NeuroD1-expressing cells were scattered within the VZ.Double immunostaining for NeuroD1 and a 0.5-hour pulse of EdU showed that NeuroD1 is expressed in basally located progenitor cells in S phase of the cell cycle (Figure 3G, arrowheads).These NeuroD1 + /EdU + cells seemed to be predominantly located in the SVZ.NeuroD1 was also clearly expressed outside of the NICD-expressing VZ (Figure 5D, arrow) and the scattered NeuroD1-positive cells within the VZ did not express NICD (Figure 5D, arrowheads), indicating the lack of NeuroD1 expression in RGs.Double staining experiments also showed that some basal progenitor cells co-expressed NeuroD1 and PH3 (not shown).These results together demonstrate that NeuroD1 is expressed in thalamic basal progenitor cells at least through S phase to M phase of the cell cycle, but not in NICDexpressing RGs. Insm1 is a zinc-finger transcription factor expressed broadly in progenitor cells within the embryonic brain and spinal cord located away from the ventricular surface [32].It is required for the generation of basal progenitor cells in the neocortex [12].We found that Insm1 is strongly expressed in a lateral band of cells within the thalamus.Comparison of Insm1 with PH3 on the same section shows that Insm1 is indeed expressed in thalamic basal progenitor cells (Figure 3C,H). Olig2 is a bHLH transcription factor expressed in the pTH-C domain of the thalamus in a rostro-ventral high to caudo-dorsal low gradient at E11.5 and E12.5 [19].We found that Olig2 is not only expressed in the VZ (Figure 3D, arrowhead) but also in a more lateral region (Figure 3D, arrow).Olig2 expression overlapped with a 0.5-hour EdU pulse (Figure 3I), and extended further laterally (Figure 3I, arrow; Figure 5E).Within the VZ, Olig2 colocalized with NICD (Figure 5E, arrowheads), suggesting that it is expressed in RGs.Thus, similar to Olig3, Olig2 is expressed in both RGs and basal progenitor cells.Olig2 also appeared to be expressed lateral to the SVZ, most likely in the mantle zone. Finally, Lhx2 and Lhx9 are LIM-homeodomain transcription factors expressed in the thalamus [33,34].In the neocortex, Lhx2 is expressed in neural progenitor cells and Lhx9 is expressed in the marginal zone [35,36].We found Lhx2/9-positive cells are largely confined outside the VZ, with only a minimum overlap with a 0.5-hour EdU pulse (Figure 3J), indicating that they are expressed mostly in postmitotic cells. Interestingly, a well-established IPC marker in the neocortex, Tbr2, a T-box transcription factor [15,29], was undetectable in the thalamus at E11.5 and E12.5 (data not shown). These results collectively show that although the thalamus has a histologically identifiable SVZ populated by basal progenitor cells and these cells share expression of some genes, such as Insm1 and NeuroD1, with neocortical IPCs, they are clearly distinct from their putative neocortical counterpart.Thalamic basal progenitor cells do not express Tbr2 and express additional markers such as Olig2 and Olig3 that are not expressed in the neocortex. ",
"section_name": "Thalamic and neocortical basal progenitor cells share some molecular properties",
"section_num": null
},
{
"section_content": "To further characterize the thalamic basal progenitor cells, we examined the expression of two bHLH proteins, Neurog1 and Neurog2, both of which are expressed in neocortical progenitor cells.In the neocortex, expression of Neurog2 is initiated soon after the division of RGs, preceding the induction of Tbr2 [15].Britz et al. [37] reported that at E12.5, 95% of Neurog1-expressing progenitor cells in the cortical VZ also express Neurog2, and at E15.5, both Neurog1 and Neurog2 are expressed in the VZ as well as the SVZ. We previously showed that Neurog1 and Neurog2 are expressed in the pTH-C thalamic progenitor domain [19].In this study, we examined the patterns of their expression in more detail.Comparison 5B, dashed line).Thus, in contrast to the neocortex, Neurog1 expression in the thalamus is confined to the VZ.Within the VZ, Neurog1 and Neurog2 showed partially overlapping but distinct expression patterns (Figure 4C).Similar to the neocortex [28], neither of these two transcription factors co-localized with NICD within the VZ (Figure 5B,C,arrowheads).This result is consistent with the hypothesis that neurogenin-expressing VZ cells are basal progenitors translocating laterally towards the SVZ.In contrast, Olig2 and Olig3 were expressed in both the thalamic VZ and SVZ and had extensive overlap with NICD within the VZ (Figure 5A,E). ",
"section_name": "Proneural bHLH proteins Neurog1 and Neurog2 are expressed in overlapping but different progenitor populations in the thalamus",
"section_num": null
},
{
"section_content": "We next examined the cell cycle properties of thalamic basal progenitor cells.First, we pulsed the progenitor cells with an S-phase marker, EdU, and analyzed the distribution of PH3-positive cells at various times after EdU injection.We detected EdU and PH3 on the same section of E11.5 and E12.5 embryos to estimate the time it takes progenitor cells to enter M phase (Figure 6).In E11.5 embryos that had been pulsed with EdU 0.5 hours prior to sacrifice, we detected a large, single cluster of EdUpositive cells that encompassed a broad medial-lateral region of the thalamic progenitor domain, suggesting the close proximity of RGs and basal progenitor cells during S phase (Figure 6A; black curve in Figure 6F,G).As expected, very few mitotic cells expressing PH3 are labeled by EdU. At 2 hours after EdU injection, we detected some EdU-positive cells at the ventricular surface and the region closer to the ventricle (Figure 6B, arrow; red curve in Figure 6F,G).Many PH3-positive cells both at the ventricular surface and in the basal location were also EdU-positive (Figure 6B, arrowheads).This indicates that, particularly at E11.5, cells start to enter M phase about 2 hours after S phase. At 4 hours, as many as 60 to 75% of PH3-expressing cells were positive for EdU at both the apical and basal locations (Figure 6C, arrowheads; Figure 6E).In addition, we found two dense clusters of EdU-positive cells that were now separated from each other.One was located close to the ventricle.The other population was located more laterally (green curve in Figure 6F,G).This separation implies a distinct migratory behavior of thalamic basal progenitor cells, which stay in the basal location from S phase to M phase.Conversely, RGs translocate their nuclei medially from S phase to M phase by interkinetic nuclear migration. At 8 hours, we again detected only a small overlap between EdU and PH3, indicating that a majority of progenitor cells labeled 8 hours before have already divided.A broad cluster of EdU-positive cells was found in the middle of the diencephalic wall (Figure 6D, between the dashed lines), and additional EdU-positive cells were found far laterally, which are likely to be postmitotic cells (blue curve in Figure 6F,G). In summary, the EdU pulse experiment distinguishes RGs and basal progenitor cells because of their distinct patterns of migration during their cell cycle. ",
"section_name": "Cell cycle properties of basal progenitor cells in the thalamus",
"section_num": null
},
{
"section_content": "By taking advantage of the EdU pulse labeling, we next examined the expression of NeuroD1, Lhx2/9, Neurog1 and Neurog2 in more detail with regard to the cell cycle status of basal progenitor cells.As already shown in Figure 3 NeuroD1 also partially co-localized with p27 (Figure 8H), a cyclin-dependent kinase inhibitor expressed in differentiating neural progenitor cells as well as postmitotic neurons [38,39], but it did not co-localize with NeuN (Figure 8C), a marker for a subset of postmitotic neurons, suggesting that NeuroD1 expression is transient. Lhx2/9 was expressed in the lateral part of the thalamus, and showed only a minor overlap with EdU at each of the pulse times (Figure 7E-H,7V).The overlap with neuronal markers NeuN and p27 was robust (Figure 8E,J), indicating that Lhx2/9 expression persists in postmitotic neurons, consistent with a previous study showing widespread expression of Lhx2 and Lhx9 in postmitotic thalamic nuclei [33]. As shown in Figure 4 8F,G) showed that neurogenins overlap with p27 but not with NeuN.Thus, the expression of neurogenins is transient. ",
"section_name": "Expression of basal progenitor markers at different stages of the cell cycle",
"section_num": null
},
{
"section_content": "In the neocortex, Neurog1 and Neurog2 together play a role in neuronal differentiation and, at the same time, in the specification of the dorsal telencephalic fate of neural progenitor cells [40].Microarray analysis shows that the expression levels of Tbr2 and NeuroD1 in the neocortex are decreased in Neurog1/2 double knockout mice [40].Although histological analysis of cortical IPCs with immunohistochemistry for Tbr2 and NeuroD1 has not been reported in these mutant mice, both PH3-positive mitotic cells and bromodeoxyuridine-labeled S-phase progenitor cells are increased in the SVZ and decreased in the VZ in Neurog2 single as well as Neurog1/2 double knockout mice [37], suggesting that these transcription factors are likely to play an important role in IPC specification and/or differentiation. In order to determine if neurogenins play a role in the formation or maintenance of basal progenitor cells in the thalamus, we analyzed Neurog1/2 double knockout mice and Neurog1 and Neurog2 single knockout mice in comparison with double heterozygous controls.We found that double knockout mice (Neurog1 -/-; Neurog2 -/-) have fewer PH3-positive, dividing basal progenitor cells in the pTH-C domain at E12.5 (Figure 9D,E).Both the absolute number and the ratio against the total PH3-positive cell number were significantly reduced from the controls.In contrast, the number of apical PH3-positive cells or the total PH3positive cells did not show a significant difference.The Neurog2 single (Neurog1 +/-; Neurog2 -/-) mutant showed reduction in absolute number of basal PH3-positive cells but not in the ratio against the total PH3-positive cells (Figure 9C,E).The Neurog1 single mutant did not show any significant difference from the control (Figure 9B,E).These results indicate that neurogenins are required for the normal number of basally dividing progenitor cells in the thalamus, and that the role of Neurog2 is only partially compensated by Neurog1. As already shown previously [41], another bHLH transcription factor, Ascl1 (also known as Mash1) is induced in the neocortex of Neurog2 single and Neurog1/2 double mutant mice.Ascl1 is normally expressed at a high level in the ventral telencephalon, suggesting a role for neurogenins in specifying dorsal telencephalic fate and suppressing ventral telencephalic fate.It has also been shown that neurogenins are required to suppress Ascl1 expression in the thalamus [41,42].Consistent with these previous findings, we found robust Ascl1 induction in the thalamus of Neurog1/2 double mutant mice (Figure 9H), whereas Neurog2 single mutants (Neurog1 +/-; Neurog2 -/-) showed much less severe induction of Ascl1 (Figure 9G).Ascl1 was not induced in Neurog1 single mutants (Neurog1 -/-; Neurog2 +/-; data not shown).These results demonstrate that neurogenins, of which Neurog2 is the prominent one, suppresses Ascl1 expression.Reduction of the basal progenitor cell number in the thalamus of neurogenin mutant mice indicates that Ascl1 does not compensate for the function of neurogenins in this cell type.Interestingly, Tbr2, a cortical IPC marker, was normally not expressed in the thalamus but was ectopically induced in the mantle zone of the thalamus of the Neurog1/2 double mutant (Figure 9K,L).Considering the fact that SVZ mitosis was increased in the neocortex [37] but decreased in the thalamus (Figure 9) of Neurog1/2 double knockout mice, we conclude that the roles of neurogenins in basal progenitor cells in the thalamus are likely different from those in the neocortex. The paired-/homeo-domain transcription factor Pax6 is known to play a critical role in thalamic development [43].As already shown in Figure 2, high-level expression of Pax6 was detected in the thalamic VZ, although the expression decreased in the rostro-ventral part of the pTH-C domain at E11.5 and later.In Pax6 mutant mice, we detected reduction of Neurog2 expression (Figure 10E, G) and ectopic induction of Ascl1 (Figure 10F,H) in the ventral part of the pTH-C domain, but not in the dorsal part (Figure 10A-D).The ratio of basal PH3-positive cells was specifically reduced in ventral sections, where a large number of Ascl1-expressing cells were intermingled with Neurog2-expressing cells (Figure 10G-I).The decrease in the number of basal PH3-positive cells was accompanied by an increase in the number of apical PH3-positive cells (Figure 10J), indicating the role of Pax6 in generating basal progenitor cells from apical progenitor cells.The total number of basal plus apical PH3-positive cells did not change between wild-type and mutant embryos, at both dorsal and ventral levels (data not shown). ",
"section_name": "Neurogenins are required for the formation and/or maintenance of basal progenitor cells in the thalamus",
"section_num": null
},
{
"section_content": "In this study, we showed that, throughout thalamic neurogenesis, a high proportion of progenitor cells divide away from the third ventricle and some of these basal cell divisions occur outside of the VZ.We found that basal progenitor cells are most abundant in the thalamic progenitor domain that expresses the bHLH transcription factors Neurog1 and Neurog2, which are also expressed in the neocortex where basal progenitor cells abound.The thalamus and the neocortex share some of the molecular markers expressed in these cell populations, including Neurog1, Neurog2, NeuroD1 and Insm1, but each also expresses a unique set of genes.For example, Tbr2 is expressed only in the neocortex and Olig2 and Olig3 are , n = 13 for Neurog1 -/-;Neurog2 +/-, n = 13 for Neurog1 +/-;Neurog2 -/-, n = 4 for Neurog1 -/-;Neurog2 -/-).One sample is one section.Numbers of basal, apical and basal plus apical PH3-positive cells as well as the ratio of basal PH3 + cells/total PH3 + cells were compared between the genotypes.One-way ANOVA for Neurog1 +/-;Neurog2 +/-, Neurog1 -/-;Neurog2 +/-and Neurog1 +/-;Neurog2 -/-embryos; F = 11.96 for basal PH3, F = 1.744 for apical PH3, F = 4.517 for basal + apical PH3, F = 5.881 for basal/total ratio.N.s., not significant; **P < 0.01.expressed only in the thalamus.We then characterized various transcription factors that are differentially expressed at different cell cycle stages of thalamic progenitor cells.We further showed that two bHLH transcription factors, Neurog1 and Neuorg2, as well as the paired-/homeo-domain transcription factor Pax6, are required for the normal number of thalamic basal progenitor cells. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Our current study has identified a domain of progenitor cells outside the thalamic VZ.We term this external zone of progenitor cells the thalamic SVZ.The term thalamic SVZ has been used before (for example, [44]), but no reports have shown the presence of progenitor cells outside of the VZ that are either dividing or in S phase of the cell cycle.In this paper, we used the presence of cleaved Notch1 (NICD) as the landmark of the VZ [28].In the neocortex, Notch signaling plays a critical role in maintaining RG fate and inhibiting the expression of Neurog2 and Tbr2, and thus the formation of IPCs [14,15,45].Within the pTH-C domain of the thalamus, progenitor cells outside the VZ start to be detectable at E11.5 and become more prominent at E12.5, although basal cell division occurs not only within the SVZ, but also in the VZ as early as E10.5, when thalamic neurogenesis has just started [25].Based on these results, we propose a classification of thalamic progenitor cells into three types based on where they divide.The first population (type I) is the RGs that divide at the apical surface of the third ventricle.If we define them as producing NICD (although the signal is weaker near the ventricular surface), they also express Olig3 and Olig2.The second (type II) and third (type III) populations divide away from the ventricular surface; type II cells divide within the VZ and type III cells divide in the SVZ.Based on our gene expression analysis, Neurog1 differentiates type II and type III cells because it is expressed by NICD-negative cells in the VZ, but not in the SVZ.Among the markers expressed in both types of progenitor cells, Neurog2 is expressed evenly between the VZ and SVZ, while Neu-roD1-expressing cells are distributed much more densely in the SVZ than the VZ.Two other bHLH factors, Olig2 and Olig3, are expressed in all three progenitor types (summarized in Figure 11).Interestingly, a recent study analyzed gene expression profiles of single progenitor cells in E14 mouse cortex and classified the progenitor cells into three clusters that likely correspond to RGs, VZ basal progenitors and SVZ basal progenitors [45].Since it is unclear whether the two basal progenitor cell clusters in the cortex represent the difference in their state of cell cycle phase (for example, G1/S phases for VZ basal progenitor cells and G2/M phases for SVZ progenitor cells) or the location of mitosis, it remains to be determined how our two progenitor populations (types II and III) differ in overall gene expression profiles and how they are related with regard to cell lineage. What is the significance of the large number of basal progenitor cells in the thalamus? Our current study shows that the thalamus is one of the brain regions where basal progenitor division is most prominent.In comparison with a similar quantification study, the ratio of basal PH3-positive cells in E12.5 thalamus is comparable to the peak ratio of basal divisions in neonatal cortex [46].As in the neocortex, basal progenitor divisions occur in both the VZ and SVZ.Although basal division of neural progenitor cells has been described in many regions in the central nervous system, including the spinal cord and the hindbrain [5][6][7][8][9], the existence of the embryonic SVZ, which is populated exclusively by basal progenitor cells and not by RGs, has been described only for the neocortex and the ventral telencephalon, where basal progenitor cells are dominant and a large number of neurons are generated.We propose that the thalamus belongs to this group of brain regions.Considering the extensive interconnections between the mammalian thalamus and the neocortex, it is intriguing to speculate that these two brain regions have evolved together to produce a balance in the large numbers of neurons needed to connect these regions. Recent lineage tracing studies show that both Neurog1and Neurog2-expressing progenitor cells produce neurons of all thalamic nuclei that project to the neocortex [19,47].Because Neurog1 is expressed in basal progenitor cells in the VZ but not in the SVZ, whereas Neurog2 is expressed in both populations, it will be interesting to determine if the basal progenitor cells in the VZ and those in the SVZ generate different sets of neurons in each nucleus.The potentially distinct postmitotic cell fates of Neurog1-and Neurog2-expressing progenitor cells might result in specific thalamic phenotypes in Neu-rog2 single knockout mice.Seibt et al. [48] showed normal expression of Lhx2 and Gbx2, both of which are widely expressed in postmitotic thalamic neurons at E12.5, in Neurog2 single knockout mice.We have also obtained similar results in Neurog1 +/-; Neurog2 -/-mice (data not shown).Thus, analysis of later embryonic stages with nuclei-specific markers would be necessary to reveal the specific roles of Neurog2 in thalamic neurogenesis. Although our study does not provide information on how the thalamic basal progenitor cells migrate, divide and produce progeny in real time, it is possible they have similar properties to neocortical IPCs, which divide symmetrically to self renew or produce two neurons, and that thalamic basal progenitor cells contribute to the diversity and the large neuronal number of thalamic nuclei.Our analysis of gene expression combined with EdU pulsing support this hypothesis (summarized in Figure 11).It is also possible that some thalamic basal progenitor cells later generate oligodendrocytes and/or astrocytes.Future lineage tracing studies using live imaging and genetic fate mapping will be able to test these possibilities. ",
"section_name": "Medial-lateral organization of the thalamic progenitor domain",
"section_num": null
},
{
"section_content": "We observed a decreased number of basal PH3-positive cells in the thalamus of Neurog2 single and Neurog1/2 double knockout embryos.In the neocortex, the level of Tbr2 mRNA is decreased in neurogenin mutant mice [37], and in vivo over-expression of Neurog2 increased Tbr2expressing cells in the cortex 24 hours after electroporation [15].However, basal PH3-positive cells were increased at the expense of apical mitosis in the neocortex of Neurog2 single and Neurog1/2 double knockouts [37].In contrast, we saw a decrease in basal progenitor cells and an unchanged number of apical progenitor cells in the thalamus of these knockout mice.Our data suggest that Neurog2, once induced in one of the daughter cells after the radial glial division in the neocortex and perhaps in the thalamus, plays a cell-autonomous role in specifying (5).Type III cells divide basally in the SVZ and generate two neurons (6) or two type III cells (7).The actual lineage relationship between the three progenitor cell types in the thalamus is a topic of future investigation.basal progenitor fate [15].Differences in the genes regulated by neurogenins in the thalamus and neocortex and the functions of these genes may account for the different phenotypes in the SVZ of neurogenin knockout mice. There are many other regions in the central nervous system where neurogenins are expressed, but, among them, only the neocortex and the thalamus appear to have prominent populations of basally dividing cells.Conversely, the ventral telencephalon expresses Ascl1 and not neurogenins, but still contains a large number of basally dividing progenitor cells.Therefore, expression of neurogenins alone is not sufficient or absolutely necessary for a large population of basal progenitor cells.Further study is needed to determine what other molecules play a shared role in the neocortex and thalamus. Our study also showed that Pax6 mutant mice have reduced numbers of basal progenitor cells in the ventral part of the thalamus where there was a severe reduction of neurogenin expression and massive induction of Ascl1.Unlike in the neurogenin mutants, however, we saw increased apically dividing cells in this region of the thalamus.The similarities and differences between the neurogenin and Pax6 mutants indicate that although Pax6 regulates the formation of basal progenitor cells in the thalamus by regulating the normal expression of neurogenins, it may also have distinct roles in RGs, which control the balance between the apical and basal progenitor cells. ",
"section_name": "Roles of neurogenins and Pax6 in the generation/ maintenance of thalamic basal progenitor cells",
"section_num": null
},
{
"section_content": "Our study provides evidence for the presence of a prominent population of basal progenitor cells in the embryonic mouse thalamus, part of which forms the SVZ.Combined analysis of transcription factor expression and cell cycle status revealed that these basal progenitor cells may be divided into two populations: one that divides in the VZ and another that divides in the SVZ (summarized in Figure 11 as a working hypothesis).We also found that neurogenins and Pax6 are required for the formation and/ or maintenance of basal progenitor cells in the thalamus.Our study implicates the importance of this special progenitor cell population in enhancing the generation of neurons within the thalamus and may also be critical for generating neuronal diversity in this complex brain region. ",
"section_name": "Conclusions",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "Care of and experimentation on mice were done in accordance with the Institutional Animal Care and Use Committee of the University of Minnesota.Noon of the day on which the vaginal plug was found was counted as E0.5.Stages of early embryos were confirmed by morphology [49].Timed-pregnant CD1/ICR mice (Charles River) were used for gene expression analysis of wild-type mice.Pax6 mutant mice [50] were obtained from G Lanuzo and M Goudling at the Salk Institute and were kept in CD1 background.Neurog1 [51] and Neurog2 [48] mutants were established in F Guillemot's lab (National Institute for Medical Research, London), produced by J Johnson's lab, and were kept in CD1 background. ",
"section_name": "Animals",
"section_num": null
},
{
"section_content": "Axial and anatomical nomenclatures are described in [19].The two progenitor domains of the thalamus, pTH-C and pTH-R, as well as the ZLI were identified by the expression of marker genes Olig3, Ascl1 and Neurog2 [19]. ",
"section_name": "Axial and anatomical nomenclature",
"section_num": null
},
{
"section_content": "Immunohistochemistry was performed as described [19,30].Additional antibodies used were: anti-NICD (rabbit, 1:100; Cell Signaling, Danvers, MA, USA), anti-NeuroD1 (goat, 1:100; Santa Cruz, Santa Cruz, CA, USA), anti-PH3 (mouse and rabbit, 1:100; Millipore, Temecula, CA, USA) and anti-Lhx2 (goat, 1:100; Santa Cruz, sc-19344).Antibody sc-19344 appears to detect a broad postmitotic region in the thalamus at E14.5 (data not shown), consistent with the possibility that it recognizes both Lhx2 and Lhx9 [33].For NICD detection, we extended the boiling time to 10 minutes to enhance the antigen retrieval, and also used a Tyramide Signal Amplification System (Perkin Elmer, Waltham, MA, USA).Detailed protocols for the entire procedures are available upon request. ",
"section_name": "Immunohistochemistry",
"section_num": null
},
{
"section_content": "In situ hybridization was performed as described [19].Insm1 cDNA was obtained from J Garcia-Anoveros (Northwestern University). ",
"section_name": "In situ hybridization",
"section_num": null
},
{
"section_content": "EdU was dissolved at 0.5 mg/ml in PBS, and injected intraperitoneally into pregnant female mice at 10 μg/g body weight.Embryos were dissected after varying amounts of time (0.5 hours, 2 hours, 4 hours or 8 hours).EdU was detected with a protocol based on that reported in [52].For simultaneous detection of EdU and various other antigens, cryosectioned brains on slides were first treated with primary and secondary antibodies.Slides were then washed once with 1× PBS and permeabilized with 0.5% Triton, then rinsed twice with 1× PBS.EdU labeling was detected with the Click-iT EdU Imaging Kit (Invitrogen, Carlsbad, CA, USA); detection solution was applied directly to slides and incubated for 15 minutes.Slides were then rinsed and coverslipped according to our previous immunostaining protocol [19]. ",
"section_name": "Cell cycle analysis",
"section_num": null
},
{
"section_content": "Images were collected with a Nikon E800 microscope or Olympus FluoView 1000 confocal microscope and assembled by Image J (NIMH) and Photoshop CS3 or CS5 (Adobe). ",
"section_name": "Image analysis",
"section_num": null
},
{
"section_content": "For single counts of PH3-expressing cells, images of 20μm-thick frontal sections were taken with a Nikon E800 fluorescent microscope.The embryonic mouse thalamus was delineated using specific markers characteristic of the pTH-C and pTH-R domains and the ZLI [19].For each section, the thalamus was divided into 20-μm bins from the ventricular surface to the lateral surface.Counts were taken for PH3-positive cells per bin.The cell counts from all sections were summed for each half brain.The proportion of PH3-positive cells away from the ventricular surface (>40 μm or >2 bins) was calculated from the total number of PH3-positive cells in the thalamus.The average proportion of PH3-positive cells was calculated with four thalami per embryonic stage. For EdU and PH3 co-localization, images were acquired by an Olympus FluoView 1000 confocal microscope.Each section was divided into bins similar to those described above, and the proportion of PH3-positive cells that co-localized with EdU was calculated from the total PH3-positive cell count.Five to six 3-μm-thick optical slices were obtained for each field of view, and two of them were taken for cell counts. For EdU-positive cell count and co-localization of EdU and basal progenitor markers, images were acquired and analyzed as described for EdU and PH3 co-localization.However, only a portion of the thalamus (the first 200 μm from the pTH-C/pTH-R border) was analyzed. For PH3-positive cell count in Pax6 mutants, two or three adjacent sections of a 300-μm-long column of the pTH-C domain were analyzed separately for dorsal and ventral levels.The ratio of basal PH3-positive cells was calculated for each of the 14 sections counted for each genotype.In addition, the absolute numbers of PH3-positive cells per section were also counted and compared. Cell count data were analyzed and graphed using Prism 4 Software (GraphPad).A one-way ANOVA test was used to determine statistical significance, where P < 0.05 indicated significance.A post-test -the Tukey multiple comparison's test -was used to determine significance among groups.Double asterisks in indicate P < 0.01, triple asterisks indicate P < 0.001. ",
"section_name": "Cell counting of PH3-and EdU-expressing cells",
"section_num": null
}
] |
[
{
"section_content": "We thank A Hanson and M Simon for their excellent technical assistance, J Johnson (University of Texas Southwestern Medical Center) for providing neurogenin knockout mouse embryos and comments on the manuscript, P Kofuji and Vision Core Facility of University of Minnesota (P30 EY011374) for the use of a confocal microscope, and Tim Cherry (Harvard Medical School) for the EdU detection protocol.Part of this research was supported by NINDS (R01 NS049357 to YN).LW was supported by University of Minnesota Undergraduate Research Opportunities Program (UROP), and KKB was supported by NICHD training grants (T32HD007480, T32HD060536) and University of Minnesota Graduate School.We thank S McLoon, P Letourneau and T Vue for comments on the manuscript and members of Nakagawa Lab for discussion. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Authors' contributions LW carried out experiments on wild-type embryos and drafted the manuscript.KKB carried out experiments on neurogenin and Pax6 mutants and drafted the manuscript.LD generated and genotyped neurogenin mutant embryos.YN conceived the study, and participated in its design and coordination and wrote 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 LW carried out experiments on wild-type embryos and drafted the manuscript.KKB carried out experiments on neurogenin and Pax6 mutants and drafted the manuscript.LD generated and genotyped neurogenin mutant embryos.YN conceived the study, and participated in its design and coordination and wrote 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
}
] |
10.1371/journal.pone.0013578
|
Identification of Epidermal Pdx1 Expression Discloses Different Roles of Notch1 and Notch2 in Murine KrasG12D-Induced Skin Carcinogenesis In Vivo
|
The Ras and Notch signaling pathways are frequently activated during development to control many diverse cellular processes and are often dysregulated during tumorigenesis. To study the role of Notch and oncogenic Kras signaling in a progenitor cell population, Pdx1-Cre mice were utilized to generate conditional oncogenic Kras(G12D) mice with ablation of Notch1 and/or Notch2.Surprisingly, mice with activated Kras(G12D) and Notch1 but not Notch2 ablation developed skin papillomas progressing to squamous cell carcinoma providing evidence for Pdx1 expression in the skin. Immunostaining and lineage tracing experiments indicate that PDX1 is present predominantly in the suprabasal layers of the epidermis and rarely in the basal layer. Further analysis of keratinocytes in vitro revealed differentiation-dependent expression of PDX1 in terminally differentiated keratinocytes. PDX1 expression was also increased during wound healing. Further analysis revealed that loss of Notch1 but not Notch2 is critical for skin tumor development. Reasons for this include distinct Notch expression with Notch1 in all layers and Notch2 in the suprabasal layer as well as distinctive p21 and β-catenin signaling inhibition capabilities.Our results provide strong evidence for epidermal expression of Pdx1 as of yet not identified function. In addition, this finding may be relevant for research using Pdx1-Cre transgenic strains. Additionally, our study confirms distinctive expression and functions of Notch1 and Notch2 in the skin supporting the importance of careful dissection of the contribution of individual Notch receptors.
|
[
{
"section_content": "Conditional tissue-specific modulation of genes using Cre/loxP recombination in genetically engineered mice provides an enormous leap forward to study gene function in detail yet requires detailed knowledge of gene regulation and expression patterns.For pancreatic targeting of genes, Pdx1-Cre mice are commonly used [1][2][3], in which Cre-recombinase is expressed under a 4.5 to 5.5 kb fragment of the Pdx1 promoter.The transcription factor Pdx1 (pancreas and duodenum homeobox gene 1) directs pancreatic cell formation, maintenance and function.Pdx1 is expressed in the region of the endoderm that ultimately gives rise to stomach, pancreas and duodenum and its function is critical for posterior foregut development [4].Postnatally, Pdx1 is mainly expressed in insulin-producing endocrine cells of the pancreas.Ablation of Pdx1 results in defects of different cell types including malformations of the pylorus and duodenum, absence of Brunner's glands and reduced numbers of specific enteroendocrine cell types in the stomach and intestine.Loss of Pdx1 function results in pancreatic agenesis, while heterozygous expression leads to defects in glucose homeostasis.Pdx1-deficient mice survive up to 6.5 days after birth, are severely dehydrated, have no fur and a delicate, cracking skin.[5][6][7][8].Here, we report epidermal PDX1 expression observed due to an unexpected skin tumor formation in Pdx1-Cre mice with activation of oncogenic Kras G12D and loss of Notch1 but not Notch2. Notch proteins are evolutionarily conserved large transmembrane receptors, which upon ligand binding undergo proteolytic cleavage mediated by the c-secretase-presenilin complex releasing the intracellular fragment (NIC).NIC is translocated to the nucleus where it binds and activates the mammalian repressor RBP-Jk thereby regulating fetal and postnatal cell fate decisions and differentiation processes [9].Notch receptors are expressed in the skin, although their precise functions remain uncertain (reviewed in [10,11]).Gain-and loss-of-function studies have suggested various functions for Notch including proliferation control, differentiation switch of developing epidermis and formation of hair follicles [12][13][14][15][16][17].Mice with epidermal loss of Notch1 as well as Presenilin-deficient mice develop epidermal hyperplasia and skin cancers [14,18].Of note, most studies have focused on Notch1 and downstream signaling members such as Rbpj or Hes1 [19,20].Very little is known about the function of Notch2 and other receptors in skin physiology and carcinogenesis.Here, we investigate the role of Notch1 and Notch2 using two different Cre expression systems.Our results provide evidence for different roles of Notch1 and Notch2 in skin development and carcinogenesis. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "Notch1 but not Notch2 deletion increases susceptibility to Kras G12D induced skin carcinogenesis in Pdx1-Cre mice To analyze the effect of Notch1 and Notch2 deficiency during pancreas carcinogenesis, we crossed previously described Pdx1-Cre [2], Notch1 fl/fl [21], Notch2 fl/fl [22] and Kras +/LSL-G12D [3] mice for generation of Pdx1-Cre;Kras +/LSL-G12D , Pdx1-Cre;Kras +/LSL-G12D ;Notch1 fl/fl and Pdx1-Cre;Kras +/LSL-G12D ;Notch2 fl/fl mice (referred to as Pdx1-Cre;Kras, Pdx1-Cre;Kras;N1ko and Pdx1-Cre;Kras;N2ko, respectively).These mice were born at the expected Mendelian ratio and successful recombination of the floxed loci in the pancreas was confirmed by PCR (Fig. 1C).Surprisingly, Pdx1-Cre;Kras;N1ko mice developed focal skin hyperplasia at 10-15 days of age and as early as 4 weeks of age developed massive skin papillomas (Fig. 1D).These lesions and tumors showed recombination of the floxed loci (Fig. 1C) pointing to epidermal Cre expression, which was further corroborated using Pdx1-Cre;Kras;N1ko;ROSA26R-LacZ reporter mice (Fig. 1F) [23].The penetrance of the skin papilloma development was 78%.In contrast, Pdx1-Cre;Kras;N2ko mice rarely developed any skin phenotype.However, double Notch1 and Notch2 knockout mice (Pdx1-Cre;Kras;N1ko;N2ko) featured an accelerated skin tumor formation (Fig. 1A andB) suggesting an essential role of Notch1 ablation in epidermal lesion development and a promoting role of Notch2 deletion.Pdx1-Cre;Kras mice manifested a skin phenotype with low penetrance, which has been observed previously [3,24].Most tumors encountered in Pdx1-Cre;Kras;N1ko mice were benign papillomas but often grew large and ulcerating, requiring euthanasia of animals for ethical reasons.Hence, the intended pancreatic carcinogenesis study was inconclusive (data not shown). Pdx1-Cre;Kras;N1ko mice developed the following skin pathologies: squamous papillomas involving the ear, neck, lips, anal and vulvo-vaginal skin, epidermal cysts, and sebaceous gland hyperplasia and cutaneous horns to lesser extend (Fig. 1D andE).Moreover, 32% of the animals developed squamous cell carcinomas (SCC), (Fig. 1E), supporting the previous observations that papillomas progressing to SCC are a common manifestation of activated Ras signaling [25][26][27].Mice without oncogenic Kras G12D but ablation of Notch1 and Notch2 (Pdx1-Cre;N1ko, Pdx1-Cre;N2ko) only very rarely developed skin abnormalities (not shown). ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "The observation that Pdx1-Cre;Kras;N1ko mice develop skin neoplastic lesions with high penetrance and undergo Cre-mediated recombination are evidence of Cre expression in the epidermis possibly due to Pdx1-Cre transgene misexpression or physiological PDX1 expression in the skin.To test both hypotheses, immuno-histochemical expression analysis was performed in the skin of wildtype and Pdx1-Cre mice, which showed a small subset of PDX1 + cells (Fig. 2A).Thus, the observed phenotype is due to physiological PDX1 expression in the skin rather than transgenic misexpression of Cre recombinase. Immunofluorescent staining of PDX1 shows that the intensity of staining was comparable to that in the duodenum and much lower than in pancreatic islet cells (Fig. 2Bi and ii).Double immunofluorescent staining revealed that PDX1 co-localizes with Kera-tin10 (K10) in the spinous layer of the epidermis (Fig. 2Biii; arrowheads).Noteworthy, a very small fraction of PDX1 + cells was located in the basal layer of the epidermis suggesting that PDX1 expression may be initiated also in this layer (Fig. 2Bi and iii; arrows). Above-mentioned experiments demonstrate that PDX1 is predominantly present in differentiated keratinocytes of the skin.To test whether PDX1 expression is regulated during keratinocyte differentiation we induced terminal differentiation in cultured wildtype keratinocytes by calcium as described [28].As early as 12 hours after calcium addition growth arrest and a switch in keratin expression occurred.As expected, treated keratinocytes showed a three-fold induction of the differentiation markers Keratin10 and Loricrin and a five fold reduction of p63 associated with amplifying keratinocytes in the basal layer of the epidermis.In addition, we found a robust 10-fold induction of Pdx1 transcript expression in treated keratinocytes (Fig. 2C).These findings strongly support the hypothesis that Pdx1 is predominantly expressed in suprabasal layers of the epidermis (Fig. 2D). Mosaic epidermal Cre expression in Pdx1-Cre mice Physiological PDX1 expression in the epidermis does not explain the stochastic character of papilloma formation in the Pdx1-Cre;Kras,N1ko mice.Hence, we speculated that Cre expression has a mosaic character or alternatively may be induced by mechanical skin irritation.To address the first hypothesis we examined X-Gal expression in Pdx1-Cre;ROSA26R-LacZ reporter mice [23].Consistent with previous studies, we found that Pdx1-Cre mice showed a mosaic recombination pattern in the pancreas [1] (Fig. 3Ai).Interestingly, similar mosaic staining was observed in the skin (Fig. 3Aii).Microscopic evaluation of X-Gal positive areas indicated that suprabasal keratinocytes underwent recombination (Fig. 3Aiii; arrowheads), supporting the hypothesis that PDX1 is mainly expressed in differentiated keratinocytes.However, we found sporadically X-Gal + keratinocytes residing in the basal layer (Fig. 3Aiii; arrow).All examined skin hyperplasia had X-Gal + basal layer cells suggesting that neoplastic structures originate from the basal keratinocytes of the skin (Fig. 3Aiv; arrow). To further asses the scale of recombination in the basal layer (K14 + ) and the spinous layer (K10 + ) of the epidermis we tested freshly isolated keratinocytes from Pdx1-Cre;N1ko mice.Cells were fractioned for K14 and K10 expression respectively using fluorescent activated cell sorting (FACS).Cre-mediated recombination was measured using quantitative PCR amplifying the recombined allele of floxed Notch1 that was normalized to input and then compared to fully recombined DNA.We found that only 5% of DNA isolated from total keratinocytes underwent recombination in Pdx-Cre;N1ko mice and most of them were found in the suprabasal layer.We sporadically (below 0.5%) found K14 + cells with recombined Notch1 loci hypothesizing that these cells could be the cell-of-origin for papilloma development (Fig. 3B). As papilloma development in Pdx1-Cre mice usually occurred in regions susceptible to grooming, scratching and wounding, we speculated that PDX1 expression may be induced in wounded skin triggering Cre-mediated Kras G12D activation and Notch1 ablation.To test this hypothesis, wounds were induced on the back skin of wild type mice.Six days after wound formation mice were sacrificed and sections of scared skin were dissected and analyzed.Increased PDX1 expression was found in the scar tissue and in the transition zone between normal and wounded epidermis (Fig. 3C).PDX1 staining pattern was nuclear and partially cytoplasmic as previously described [29][30][31][32].Quantitative RT-PCR indicated a three-fold induction of Pdx1 and highly increased Keratin6 transcript levels in wounded compared to normal epidermis (Fig. 3D) supporting PDX1 expression in wounded skin.In summary these results denote (i) physiological Pdx1 expression in the skin, (ii) restricted to differentiated keratinocytes but sporadically present in K14 + basal cells, (iii) mosaic Pdx1-Cre epidermal expression, and (iv) Pdx1 induction in wounded skin. Histopathology of skin tumors developing in Pdx1-Cre;Kras;N1ko mice Histological investigations revealed that the papillomas and hyperplastic epithelium cover thin expansions of a fibroblastic stroma often with mild chronic inflammatory infiltrates.Local hyperplasia and squamous papillomas were well differentiated, rarely demonstrating focal dysplasia (Fig. 1E).Sections of typical papillomas were analyzed by immunofluorescence for differentiation markers including Keratin 14, 10 and Loricrin.In the papillomas all three keratins were expressed in a manner similar to normal skin, except that there was a delay in the onset of K10 expression consistent with an expansion of the proliferative compartment expressing K14 and CyclinD1 (Fig. 4).In line with the hyperplastic character was the expression of K6, a keratin usually expressed in hair follicles or in pathological conditions resulting in hyperplasia (Fig. 4).The observed keratin expression pattern is characteristic of well-differentiated squamous papillomas.Older mice developed hyperproliferative lesions that exhibited cellular atypia, increased mitosis and an invasive growth pattern with characteristic keratin 'pearls' formation and a high degree of keratinization that are diagnostic of well-differentiated SCC.Of note, no basal cell carcinomas (BCC) were observed in Pdx1-Cre;Kras;N1ko mice and no signs of a metastatic disease were observed. Immunohistochemical characterization of papillomas revealed strong activation of Ras-dependent phospho-ERK consistent with previous studies [33] as well as robust MYC expression associated with skin neoplastic transformation [34].Interestingly, robust p63 expression throughout the papilloma tissue was noted.Normally, the presence of p63 is restricted to the thin layer of basal keratinocytes due to inhibition by Notch1.Expression of p63 is characteristic for progenitor and multiplying cells of the epidermis.Expanded and strong CyclinD1 staining supports this conclusion (Fig. 4).This expression pattern is common and characteristic for cutaneous neoplasia. ",
"section_name": "Evidence of Pdx1 expression in vivo and in vitro",
"section_num": null
},
{
"section_content": "Although the role of Notch receptors in the skin has already been intensively studied [12][13][14][15][16][17], we aimed to characterize epidermal Notch1 and Notch2 deficiency in our model.To do so, Notch1 fl/fl [21] and Notch2 fl/fl [22] mice were crossed with basal keratinocyte-specific Keratin5-Cre mice [35] (named K5;N1ko and K5;N2ko respectively).These mice were born at the expected Mendelian ratio (Fig. 5B) and successful recombination of the floxed loci was confirmed in isolated primary keratinocytes by immunoblot (Fig. 6A). Consistent with previous studies, K5;N1ko mice did not develop proper hair follicles showing a 'naked' phenotype.Additionally, the epidermis was thinner, easily cracking and prone to injury (Fig. 5B, D andE).Such a phenotype has been attributed to a role of Notch1 in the stimulation of keratinocyte differentiation [19,36,37].Before the age of 9 months, K5;N1ko mice developed extensive hyperplasia and keratinization of the corneal epithelium, which resulted in opaque plague formation and blindness (Fig. 5B andC) [14].All analyzed mice (n = 4) developed skin neoplasia at 9 to 12 months of age and additionally BCC, SCC and papillomas were noticed (Fig. 5B andC).By contrast, K5;N2ko mice featured a non-pathological skin and hair follicle formation (Fig. 5B andD) with normal growth cycles.However, impairment of hair growth direction that manifested in more upwards-ruffle appearance of fur was observed (Fig. 5B).Mice followed up to 12 months of age (n = 4) did not show any sign of tumorigenesis.Taken together, our findings confer that Notch1, but not Notch2 is a tumor suppressor and plays a crucial role in proper skin development and differentiation. Since expression in different compartments may explain distinct Notch1 and Notch2 functions, we analyzed the expression pattern of these receptors using immunohistochemical staining as well as transgenic Notch1-GFP [38] and Notch2 lacZ knockin [39] reporter mice.We found Notch2 and X-Gal as a surrogate for Notch2 expression in spinous and granular layers of the epidermis (Fig. 5A).Notch1 and GFP expression in Notch1-GFP mice was found throughout the epidermal layers as previously described [37], including the basal layer of keratinocytes formed by stem cells and highly proliferative transit amplifying cells (Fig. 5A).Besides these differences in expression, different and contextspecific functions of Notch1 and Notch2 have been described.We thus isolated and cultured primary keratinocytes from K5;N1ko and K5;N2ko mice, which showed no protein expression of the respective Notch receptor (Fig. 6A) and significantly downregulated levels of Hes1 transcripts (Fig. 6B) Notch1 signaling is essential for proper skin differentiation through induction of p21 (WAF1/Cip1) [37,40].We speculated that Notch2 signaling might not be required for this process since it is expressed mainly by differentiated keratinocytes.p21 is a cyclin-dependent kinase inhibitor that induces cell cycle arrest [41], predictably its loss is commonly associated with skin malignancies, particularly in an active Ras context [34].We found that p21 expression was highly reduced in Notch1 ablated cells whereas no significant differences were noted in Notch2 deficient keratinocytes both on mRNA and protein level (Fig. 6A, Figure 2. Pdx1 is physiologically expressed in the adult mouse epidermis.A: Immunohistochemical PDX1 staining of normal wildtype epidermis (i, ii) reveals that PDX1 is expressed in suprabasal keratinocytes (black arrowheads) and only rarely in basal cells (black arrows).Pdx1-Cre;Kras;N1ko papilloma (iii) is strongly positive for PDX1.Inclusion (iii) shows positive staining of pancreatic islet cells.Nuclei were contrastained with methyl green (i, ii) or hematoxilin (iii).B: Immunofluorescent PDX1 staining (i) indicates positive keratinocytes in the suprabasal (white arrowheads) and the basal (arrow) layer of the skin.Signal strength is comparable to that in duodenum cells (ii, arrowheads) and weaker than in pancreatic islet cells (ii, inclusion).Double immunofluorescence (iii) demonstrates that the majority of PDX1 + cells co-localize with a suprabasal marker Keratin10 (arrowheads) however, a small subset of PDX1 + cells can be found in the basal layer of the epidermis (arrow).Asterisks indicate unspecific staining of stratum corneum.C: Pdx1 expression in cultured keratinocytes is increased during Ca ++ -induced differentiation.Quantitative RT-PCR of Pdx1, Keratin10, Loricrin and p63 transcripts in induced primary keratinocytes in vitro.D: Schematic representation of PDX1 expression in the epidermal layers: (SC) Stratum Corneum, (GL) Granular Layer, (SL) Spinous Layer, (BL) Basal Layer, (BM) Basement Membrane, (D) Dermis and their markers: Loricrin, K1/10, K5/14.The scale bars represent 50 mm.doi:10.1371/journal.pone.0013578.g002B).These results support the hypothesis that p21 is mainly regulated by Notch1 but not by Notch2 potentially due to cell-and context-specific differences. ",
"section_name": "Notch1 but not Notch2 is a tumor suppressor in the skin",
"section_num": null
},
{
"section_content": "As an increased level of active b-catenin is commonly associated with skin malignancies [18,42,43], we investigated the regulation of this pathway in Notch1 and Notch2 ablated epidermis. Immunohistochemical analysis revealed increased levels of nuclear localized b-catenin (active b-catenin) in K5;N1ko mice in agreement with previous studies [14].Remarkably, neither wildtype nor K5;N2ko mice showed strong epidermal active bcatenin staining (Fig. 5D).Furthermore, immunoblot analysis of primary keratinocytes isolated from K5;N1ko and K5;N2ko mice exhibited a similar pattern (Fig. 6A). Differences in expression of Notch1 and Notch2 in the epidermal layers as well as receptor-specific regulatory mechanisms may contribute to distinct and potentially tumorigenic alterations of b- catenin activity.Therefore, we examined the capabilities of active Notch1 (N1IC) and Notch2 (N2IC) to inhibit b-catenin signaling activity in primary keratinocytes using a luciferase reporter assay.Both Notch receptors were able to inhibit b-catenin activity but N1IC was a significantly stronger inhibitor.Forced expression of N1IC represses b-catenin signaling by over 90% whereas N2IC overexpression leads only to a modest reduction of 30% (Fig. 6C).At the same time both Notch receptors showed a similar induction of Hes1 promoter activity, serving as a read-out for similar activation of canonical Notch signaling (Fig. 6 C). Taken together, these results support a context-and cell-specific function in addition to a distinct expression pattern of Notch and Notch2 in keratinocytes. ",
"section_name": "Notch1 but not Notch2 is a suppressor of b-catenin in the skin",
"section_num": null
},
{
"section_content": "Neoplasms originating from cutaneous epithelial cells are the most common cancer-type in the United States with an annual incidence of over 1 million cases [44].Developmental signaling pathways play a key role in the induction and progression of cancer.Our study reports a previously unrecognized epidermal expression of PDX1 and adds further evidence for a pivotal role of Notch1 but not Notch2 as a tumor suppressor in the skin, which may be particularly interesting in the light of new therapeutic approaches targeting single Notch receptors [45,46]. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "As PDX1 is mainly expressed in the pancreas and duodenum, the Pdx1 promoter is commonly utilized for pancreas-specific transgenic mouse lines.Surprisingly, we found conditional gene deletion in the skin using a Pdx1-Cre strain [2].Further research provided strong evidence that PDX1 is physiologically expressed in the suprabasal layers of the skin (Fig. 2A andB; arrowheads) and rarely in basal keratinocytes (Fig. 2A andB; arrows).A similar pattern of Pdx1 expression was observed in differentiation induced cultured keratinocytes (Fig. 2C).This hypothesis is supported by reports indicating a skin phenotype of Pdx1 knockout mice, which survive 6.5 days postpartum and have, among other characteristic features, thin and cracking skin with little or no fur [7].While these skin abnormalities may be due to indirect effects, they suggest a role of PDX1 during skin development, which should be addressed in further studies, e.g. by analyzing keratinocyte-specific Pdx1 knockout mice, which however is beyond the scope of this report. In contrast to the ubiquitous expression of Pdx1 in the suprabasal layers of the skin, Pdx1-Cre;Kras,N1ko mice developed skin papillomas and other cutaneous lesions only in preferred sites suggesting that Cre-mediated recombination may be mosaic and/or occurs in the cells resistant to neoplastic transformation.Notably, Cre expression in Pdx1-Cre mice is mosaic such that Cre-mediated recombination occurs far less frequently as would be suggested by the observed PDX1 expression.In addition, papillomas and most other skin tumors typically originate from the basal layer; in fact development from the suprabasal layer is a rather unlikely scenario (Fig. 7).Although PDX1 is mainly expressed in the suprabasal keratinocytes, we occasionally found PDX1 expression and Cremediated recombination in K14 + cells (Fig. 3A, B and7).These observations may be the reason for the relatively few tumors developing per animal.Interestingly, tumors of Pdx1-Cre;Kras,N1ko mice usually develop around exposed areas of the skin (Fig. 1D), possibly due to Pdx1 activation in wound and scar associated basal layer keratinocytes (Fig. 3C).We speculate that cutaneous aggravation or micro-wounds due to grooming and scratching may trigger an inflammatory reaction and wound healing processes with upregulated Pdx1 and Notch expression [47], thus forming a tumor-prone environment in Pdx1-Cre;Kras;N1ko mice. Intriguingly, other studies have reported skin phenotypes using Pdx1-Cre mice despite the fact that different transgenic strains were utilized [3,24].These reports support our finding that Pdx1 is expressed in the skin.However, only defined genetic alterations lead to a cutaneous phenotype.In the most often analyzed Pdx1-Cre;Kras mouse model, skin lesions were only rarely observed (below 5%, Fig. 1B and [3,24]).In our study, Pdx1-Cre;Kras;N1ko but not Pdx1-Cre;Kras;N2ko or Pdx1-Cre;Kras developed skin lesions (Fig. 1A andB) which points to the importance of Notch1 but not Notch2 for skin tumor development. ",
"section_name": "Epidermal PDX1 expression",
"section_num": null
},
{
"section_content": "Different Notch receptors have often distinct expression patterns, ligand preferences and discrete downstream signaling.Although different Notch receptors can compensate each other e.g. in pancreas development [48], individual Notch receptors commonly have distinct functions in development [49], tumorigenesis [46,[50][51][52] or tissue regeneration [53].The result of this study points to differences in expression pattern and distinctive cellular effectors as main cause of the diverse Notch1 and Notch2 knockout phenotypes.First, we found that Notch1 and Notch2 are present only in partially overlapping layers of the epidermis.Consistent with previous studies, Notch1 is present throughout all skin layers including the tumor-prone basal layer of the skin, whereas Notch2 is expressed exclusively in suprabasal keratinocytes [37].These findings were confirmed using immunohistochemical staining as well as Notch1-GFP and Notch2 LacZ reporter mice (Fig. 5A).This divergent expression pattern is very likely at least partially responsible for the downregulation of p21 in Notch1but not Notch2-deficient keratinocytes and in line with previous studies [37,40].p21 is a cyclin-dependent kinase inhibitor that induces cell cycle arrest [35] and its loss is commonly associated with skin malignancies, particularly in an active Ras context [36].In Kras G12D -induced tumorigenesis inhibition of p21 via Myc activation, observed in Pdx1-Cre;Kras;N1ko papillomas (Fig. 4), is a critical step for malignant transformation [34].Thus, the observed differences in p21 induction by Notch1 and 2 receptors (Fig. 6A andB) could partially explain the observed phenotypes. The second notable difference between Notch1 and Notch2 was their ability to inhibit b-catenin-mediated signaling.b-catenin is responsible for hair-follicle morphogenesis and epidermal stem cell maintenance [54], whereas the disruption of the b-catenin signaling has been associated with several malignancies of the skin [18,42,43].Notch1 deficiency leading to accumulation of bcatenin in the nucleus has been associated with tumorigenesis [14].Surprisingly, we did not observe a similar effect when the Notch2 receptor was abrogated (Fig. 5D and6A).Additionally, we provide in vitro evidence of different inhibition capacities between both receptors (Fig. 6C) further supporting the postulate of distinct molecular functions of Notch1 and Notch2. In line with the non-redundant roles of Notch1 and Notch2 in keratinocytes is the accelerated papilloma formation in double Notch1/2-deficient mice (Fig. 1A andB), suggesting that Notch2 cannot fully compensate for Notch1 loss.Besides different roles in regulation of p21 and b-catenin, Notch expression dosage may play a role as was recently shown [17].In this study Notch1 loss promoted skin tumorigenesis in a non-cell autonomous manner by impairing skin-barrier integrity and creating a wound-like microenvironment in the epidermis.Of note, Notch2 ablation alone had no such capabilities unless combined with a Notch3 knockout, suggesting that a certain threshold of Notch signaling is essential for skin homeostasis. In conclusion, our results provide strong evidence for epidermal expression of Pdx1 as of yet not identified function as well as distinctive roles of Notch1 and Notch2 in skin tumorigenesis potentially via different p21 and b-catenin pathway modulation. ",
"section_name": "Notch1 and Notch2 play different roles in skin tumorigenesis",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "Kras +/LSL-G12D , Notch1 fl/fl , Notch2 fl/fl , Pdx1-Cre and Keratin5-Cre transgenic mice have been described before [2,21,22,27,35].Mice were interbred to obtain Pdx1-Cre;Kras +/LSL-G12D (Pdx1-Cre;Kras), Pdx1-Cre;Kras +/LSL-G12D ;Notch1 fl/fl (Pdx1-Cre;Kras;N1ko), Pdx1-Cre;Kras +/LSL-G12D ;Notch2 fl/fl (Pdx1-Cre;Kras;N1ko), Keratin5-Cre;Notch1 fl/fl (K5N1ko) and Keratin5-Cre;Notch2 fl/fl (K5N2ko) mice.Previously described reporter strains LSL-ROSA26R-LacZ, Notch1-GFP and Notch2 lacZ [23,38,39,55], were used as indicated in the text.All animals were of mixed C57BL/6J;129SV background.Animal care and experimental protocols were conducted in accordance with German animal protection laws and approved by the Institutional Animal Care and Use Committee at the Technical University of Munich. ",
"section_name": "Mouse strains",
"section_num": null
},
{
"section_content": "Kaplan-Meier curves were calculated using the tumor free survival time for each mouse from all littermate groups.The logrank test was used to test for significant differences between the four groups.For gene expression analysis the unpaired two-tailed Student's t-test was used.For P values the following scale was used: * p,0.05, ** p,0.01, *** p,0.001. ",
"section_name": "Statistical Analyses",
"section_num": null
},
{
"section_content": "For morphologic, immunohistochemical, and immunofluorescence studies specimens were fixed in 4% buffered formalin then processed as described previously [56] and embedded in paraffin.Tissues were sectioned 4 mm and stained with hematoxylin and eosin (HE) or used for immunohistochemical studies with antibodies: CDK4 (Santa Cruz Biotechnology), K14, K10, K6, Loricrin (Covance), Notch1 (Abcam), Notch2 (The Developmental Studies Hybridoma Bank), pERK, (Cell Signaling), p63, CyclinD1 (BD), active-b-catenin (Upstate), PDX1 (gift of C.V. Wright).X-Gal staining of cryosections (10 mm) was carried out according to standard protocol, counterstained with nuclear fast red.Immunofluorescence was performed using Alexa 488 and 555 (Invitrogen).Nuclei were stained with DAPI.Pictures were taken using an Axiovert 200 M fluorescence inverse microscope equipped with the Axiovision software (Zeiss). ",
"section_name": "Histology and Immunohistology",
"section_num": null
},
{
"section_content": "HE stained sections were evaluated by a pathologist (B.S.) with expertise in human and mouse cancer pathology.The pathologist, where needed, also reviewed immunohistochemical stainings. ",
"section_name": "Histopathological Evaluation",
"section_num": null
},
{
"section_content": "Protein extracts from freshly isolated primary keratinocyte cells were obtained using RIPA buffer containing proteinase inhibitors -Complete (Roche).Lysates were separated on standard SDS-PAGE electrophoresis, transferred to PDVF membranes as described previously [56] and incubated with antibodies: b-actin (Sigma), Notch1 (BD Pharmigen), Notch2 (The Developmental Studies Hybridoma Bank), p21 (LabVison), active b-catenin (Upstate).Antibody binding was visualized using horseradish peroxidase-labeled secondary antibodies and ECL reagent (Amersham). ",
"section_name": "Western Blot Analysis",
"section_num": null
},
{
"section_content": "Keratinocytes were isolated from 3 to 4 week old mice as described previously [57].Briefly, mice in anlagen phase were sacrificed, trunk skin was removed disinfected and enzymatically treated to allow separation of epidermis from dermis.Detaching keratinocytes were collected, filtered through Teflon mesh (100 mm), washed and plated on Petri dish previously coated with collagen and fibronectin.Cells were maintained in DMEM Spiner modification media (Sigma) with addition of 8% FCS treated with Chelex (BioRad), 10 mg/ml Transferrin, 5 mg/ml Insulin, 10 mM Phosphoethyloamine, 10 mM Ethyloamine, 0.05 nM CaCl 2 (Sigma), 10 ng EGF, 0.36 mg/ml Hydrocortisone (Chemicon), 1% Glutathion, 1% Pen/Strep (Invitrogen). Keratinocytes were plated and cultured for 3 to 5 days before use in luciferase and differentiation assays.Growth medium was changed every day.Induction of keratinocyte differentiation was achieved by addition of CaCl 2 to final concentration of 1.2-2 mM. ",
"section_name": "Primary Keratinocytes Culture",
"section_num": null
},
{
"section_content": "Total isolated keratinocytes were stained with K14 or K10 antibodies (Covance) for 1 h at 4uC.Cells were washed in PBS +1% BSA and stained with the secondary antibody Alexa 488 (Invitrogen).Keratinocytes were washed and stained with propidium iodide followed by sorting using a FACS Aria 2 (BD Bioscience).DNA was isolated from the sorted cells utilizing DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer's instructions.Recombination of genomic DNA was quantified by qPCR using the following program: 95uC for 10 min, 35 cycles of 95uC for 10 sec, 62uC for 10 sec and 72uC for 30 sec on a LightCycler 480 (Roche).All samples were analyzed in triplicate. ",
"section_name": "Fluorescent Activated Cell Sorting for Cre-mediated recombination analysis in Keratinocytes",
"section_num": null
},
{
"section_content": "A luciferase reporter assay was performed with a pair of luciferase reporter constructs TOPFLASH, containing three copies of the TCF/LEF binding sites and FOP-FLASH, containing mutated binding sites (Upstate Biotechnology).Primary keratinocytes were cultured in 6-well plates and transiently transfected in triplicates with Fugene 6 (Roche) and TOP/FOP or Hes1-luc plasmids with addition of forced expressing active Notch1 (N1IC) or Notch2 (N2IC) pcDNA3 plasmids and pRL-TK (Promega).Luciferase activity was measured with the Dualluciferase reporter assay system (Promega), with the Renilla luciferase (pRL-TK) activity as an internal control, 48 h after transfection.The experiment was repeated three times, the mean of all results was taken and expressed as a percentage of induction over control ( = 100%). ",
"section_name": "Luciferase assay",
"section_num": null
},
{
"section_content": "Skin wound healing analysis was performed as described previously [58].Briefly, full-thickness excisional skin wounds (6 mm in diameter) were made in WT mice.Animals were killed 5 days after wounding (n = 4), and an 8-10 mm area, including the complete epithelial margins, was collected and used for histopathological analysis.Three small areas (363 mm) of wounded and unaffected skin from the same animal were used to prepare RNA for expression analysis.Four mice were analyzed. ",
"section_name": "Wounding and preparation of wound tissue",
"section_num": null
},
{
"section_content": "RNA was isolated from primary keratinocytes using Qiagen RNeasy Isolation Kit followed by cDNA synthesis (SuperScript II, Invitrogen).Real-Time PCR was performed with 800 nM primers diluted in a final volume of 20 ml in SYBR Green Reaction Mix (Applied Biosystems).RT-PCRs were performed as follows: 95uC for 10 min, 45 cycles of 95uC for 10 sec, 60uC for 10 sec and 72uC for 10 sec.using LightCycler 480 (Roche).All samples were analyzed in triplicate.Cyclophilin and HPRT were used for normalization.The following primers were used: K6a-F 59-GAGCTGGCTTTGGTGGTG-39 ",
"section_name": "Quantitative RT-PCR",
"section_num": null
}
] |
[
{
"section_content": "We thank Reinhard Fa ¨ssler, Ramin Massoumi and Rudolf A. Rupec for helpful discussions and expertise help with mouse models as well as keratinocytes isolation and culture.The Notch2 antibody developed by S. Artavanis-Tsakonas was obtained from the Developmental Studies Hybridoma Bank.We thank W. Gao, Y. Hamada, C.A. Klug and J. Takeda for providing Notch1-GFP, Notch2 LacZ and Keratin5-Cre mice respectively.We are grateful to C.V. Wright for the PDX1 antibody.We thank M. Neuhofer and S. Ruberg for excellent technical assistance. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "This work was supported by grants from the German Cancer Aid (#107195), The Lustgarten Foundation (RFP05-14 and 06-12) and the German Research Foundation (SI 1549/1-1; all to J.T.S.).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: PKM JTS.Performed the experiments: PKM.Analyzed the data: PKM BMG BS RS JTS.Contributed reagents/materials/analysis tools: HN UZS FR.Wrote the paper: PKM JTS. ",
"section_name": "Author Contributions",
"section_num": null
}
] |
10.3390/cancers12071876
|
Aspartic Aminopeptidase Is a Novel Biomarker of Aggressive Chronic Lymphocytic Leukemia
|
<jats:p>Treatment of chronic lymphocytic leukemia has advanced substantially as our understanding of the kinase signal transduction pathways driven by the B cell receptor (BcR) has developed. Particularly, understanding the role of Bruton tyrosine kinase and phosphatidyl inositol 3 kinase delta in driving prosurvival signal transduction in chronic lymphocytic leukemia (CLL) cells and their targeting with pharmacological inhibitors (ibrutinib and idelalisib, respectively) has improved patient outcomes significantly. The kinase signaling pathway induced by the BcR is highly complex and has multiple interconnecting branches mediated by tyrosine and serine/threonine kinases activated downstream of the BcR. There is a high level of redundancy in the biological responses, with several BcR-signaling kinases driving nuclear factor kappa B activation or inducing antiapoptotic Bcl-2 genes. Accordingly, common gene targets of BcR-signaling kinases may serve as biomarkers indicating enhanced BCR-signaling and aggressive disease progression. This study used a gene expression correlation analysis of malignant B cell lines and primary CLL cells to identify genes whose expression correlated with BCR-signaling kinases overexpressed and/or overactivated in CLL, namely: AKT1, AKT2, BTK, MAPK1, MAPK3, PI3KCD and ZAP70. The analysis identified a 32-gene signature with a strong prognostic potential and DNPEP, the gene coding for aspartic aminopeptidase, as a predictor of aggressive CLL. DNPEP gene expression correlated with MAPK3, PI3KCD, and ZAP70 expression and, in the primary CLL test dataset, showed a strong prognostic potential. The inhibition of DNPEP with a pharmacological inhibitor enhanced the cytotoxic potential of idelalisib and ibrutinib, indicating a biological functionality of DNPEP in CLL. DNPEP, as an aminopeptidase, contributes to the maintenance of the free amino acid pool in CLL cells found to be an essential process for the survival of many cancer cell types, and thus, these results warrant further research into the exploitation of aminopeptidase inhibitors in the treatment of drug-resistant CLL.</jats:p>
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is a malignancy of mature B cells.With more than 11,000 cases diagnosed every year in Europe and 15,000 in the USA, CLL is the most common type of leukemia diagnosed in the Western world [1].CLL displays remarkable clinical heterogeneity, with many patients experiencing an indolent disease state and never requiring treatment, contrasting others who develop an aggressive disease needing treatment soon after diagnosis.Therefore, the accurate identification of patients with the aggressive disease phenotype is important. CLL is characterized by the clonal expansion of CD5 + CD23 + B cells [2], which is driven by the accumulation of cell-intrinsic aberrations, as well as by the enhancement of B cell receptor (BcR) signaling.Approximately 55-60% of CLL patients display somatic hypermutation (SHM) in the rearranged clonotypic immunoglobulin heavy variable (IgHV) genes of the BcR (mutated CLL, M-CLL), while the remainder carry IgHV genes with minimal or no SHM (unmutated CLL, U-CLL).M-CLL is generally associated with a favorable prognosis, contrasting U-CLL, which typically exhibits an aggressive clinical course and adverse prognosis [3]. Engagement of the BcR with antigens triggers a cascade of kinase activation driving the activation of B cells.Upon activation of the BcR, the first kinases activated are the spleen tyrosine kinase (Syk) and the SRC kinase Lyn, which are recruited to the BcR complex and phosphorylate its immunoreceptor tyrosine-based activation motifs (ITAMs), namely Igα (CD79a) and Igβ (CD79b) [4].Phosphorylation of Igα and Igβ at the ITAMs allows for the recruitment of adaptor proteins and further kinases, such as Bruton's tyrosine kinase (BTK) and phosphatidylinositol 3-kinase (PI3K), driving the downstream activation of the prosurvival AKT pathway, followed by mTOR (mammalian target of rapamycin), NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), and/or ERK (extracellular signal-regulated kinase) activation [4].Studies on CLL cells demonstrated that BCR signaling is enhanced by a Zeta-associated protein of 70 kDa (ZAP70).ZAP70 is a homolog of Syk, first identified as the apical kinase in the T cell receptor-mediated signal transduction pathway.The constitutive activity of ZAP70 contributes to the untethered activation of downstream signaling kinases, leading to enhanced BCR signaling [5].High levels of ZAP70 protein expression correlate with higher disease stages, i.e., Binet stage B or C [6].Expression of the ZAP70 protein is also an established surrogate marker for the IgHV gene SHM status, whereby U-CLL correlates with a 5.5-fold more ZAP70 protein concentration [6,7]. Until recently, the mainstream management of CLL consisted of a combinatorial drug regimen of genotoxic agents, such as the alkylating agent cyclophosphamide and purine analogs (fludarabine) in combination with the anti-CD20 monoclonal antibody, Rituximab.However, over time, many CLL patients developed resistance against the concurrent genotoxic and cytotoxic stresses triggered by these agents.This evolution in resistance can be abstracted to the production of trophic factors released by the lymph node microenvironment and activation of BcR-signaling kinases, resulting in compensatory prosurvival signal transduction, such as induction of the antiapoptotic BCL2 [8,9] protein family. Of the BcR-signaling kinases, the most commonly used biomarker for aggressive CLL is ZAP70.However, not all patients with a high ZAP70 expression have an aggressive disease, and, vice versa, not all patients with a low level of ZAP70 have an indolent disease.For example, in approximately 25% of patients, discordance has been observed between the ZAP70 expression and IgHV gene SHM status [10].This observed discordance can be explained by the presence of additional genetic high-risk features, such as chromosomal deletions (e.g., 11q or 17p) [10].Furthermore, while both 17p deletion and ZAP70 expression predict the aggressive disease, the 17p deletion falls within the ZAP70-negative patient group [10].Moreover, there is also a high variation in ZAP70 expression depending on where the sample was collected from within the patient, i.e., peripheral blood, bone marrow, and lymph node specimen [11]. ZAP70 enhances BcR signaling by facilitating the recruitment of other kinases to the BcR complex, such as Syk, rather than via its own kinase activity [12].Moreover, the expression of ZAP70 enhances and prolongs the activation of several key mediators of BcR signaling, such as Syk, ERK, and Akt, and decreases the rate of ligand-mediated BcR internalization.ZAP70 is also found to interact with other signaling kinases and adaptor proteins like PI3K and Shc, respectively [12].Thus, the constitutive activation of kinases like Syk, PI3K, Akt, Btk, and ERK in response to the ZAP70 expression further drives BcR signaling, enhancing prosurvival signaling and the consequent resistance against cellular stresses triggered by chemotherapeutics.Drugs targeting key BcR-signaling kinases, particularly Btk and PI3K, have shown great success in treating patients with the aggressive disease.Especially, two oral kinase inhibitors, idelalisib (PI3K inhibitor) and ibrutinib (BTK inhibitor), have changed the standard of care for CLL patients in both the relapsed and frontline settings [13].Despite the outstanding efficacy observed with these agents, patients still relapse, and some patients fail to respond, which is likely, since many patients have an increased activity of multiple BcR-signaling kinases, and, due to their complex interrelationship, the inhibition of only one of these kinases triggers a compensatory mechanism via other kinases and a consequent development of resistance. In this study, we aimed to identify common or shared targets of BcR-signaling kinases as better biomarkers of aggressive CLL and potential targets to reverse drug resistance using a gene co-expression network analysis.We hypothesize that signal transduction pathways driven by the lymphoid microenvironment-activated kinases converge on common effectors.The identification of these effectors can provide novel and early biomarkers, aggressive CLL, and possible drug targets to sensitize drug-resistant CLL cells. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "2."
},
{
"section_content": "While ZAP70 contributes to enhanced BcR-signaling via multiple mechanisms, now it is clear that additional pathways, activated by either oncogenic transformation triggering cellular stress pathways or the lymphoid microenvironment, play an important role.Thus, in order to identify biomarkers of enhanced BcR-signaling, we identified genes whose expressions correlated with multiple BcR-signaling kinases, namely: ZAP70, AKT1, AKT2, BTK, MAPK1, MAPK3, and PI3KCD, using bi-weight mid-correlation analysis.For the analysis, 14 B cell gene expression microarray datasets were obtained from ArrayExpress at the EBI (European Bioinformatics Institute) and Gene Expression Omnibus (GEO) at NCBI.A description of the studies and the number of genes and samples in the datasets are summarized in Table S1.The bi-weight mid-correlation values were first individually calculated for the 14 datasets.Then, a threshold value of 0.5 was set to select the highly correlating genes.Of these genes, there were 1262 whose expressions correlated with ZAP70 and at least one other BcR-signaling kinase in at least five datasets (Figure 1A,B).From this list, the genes that showed correlations with multiple kinases were selected out for further analysis.The final selection contained 32 genes whose expressions correlated with ZAP70 and a minimum of two other BcR-signaling kinases (Table S2).Of these 32 genes, the ones that correlated with ZAP70 and AKT1/2 expressions also correlated with MAPK3 and PI3KCD expressions but not with BTK (Figure 1C,D).Interestingly, there was a relatively small overlap between AKT1/2 and PI3KCD co-expressed genes.Many of the genes that correlated with ZAP70 and PI3KCD also showed co-expressions with MAPK3 but not with AKT1/2 or BTK, indicating that the gene expressional programs associated with different BcR-signaling kinases are diverse and only partially overlapping (Figure 1C,D). A network analysis found that 28 of the 32 genes formed a closely connected, minimal network, clustering around four main nodes: HNF4A (hepatocyte nuclear factor 4 alpha), EED (embryonic ectoderm development), ELAVL1 (ELAV-like RNA binding protein 1), and MAPK1/3 and that the 32-gene signature reports on the activity of these four genes/pathways.This well-interlinked signaling network (Figure 1E) contains nodes already known to have a role in CLL, such as EZH2 and NF-κB, and also identified new pathways not well-associated with CLL (HNF4A and ELAVL1 nodes) [14][15][16].Partial integration and diversity in gene expressional programs that drive the expressions of BCR-signaling kinases.(A) Total number of genes whose expressions correlated with that of ZAP70 and at least one other BCR-signaling kinase in at least five datasets.(B) The number of correlating genes identified for each BCR-signaling kinase.(C) Circos plot showing the distribution of common targets of BCR-signaling kinase pairs.(D) Matrix representation of the number of genes that are common correlating genes of BCR-signaling kinase pairs.(E) Interaction network of the 32 genes identified.Ingenuity pathway analysis was carried out to identify gene networks the 32 BCRsignaling kinase co-expressed genes reported on.Grey-shaded genes are the identified BCR-kinase correlating genes. ",
"section_name": "Identification BCR Signaling Kinase-Correlating Genes",
"section_num": "2.1."
},
{
"section_content": "Further analysis was directed towards validating the prognostic power of the identified genes by analyzing the time to treatment and overall survival responses using an independent transcriptomic dataset of 107 CLL patients [17].For both analyses, the hazard ratio associated with Ingenuity pathway analysis was carried out to identify gene networks the 32 BCR-signaling kinase co-expressed genes reported on.Grey-shaded genes are the identified BCR-kinase correlating genes. ",
"section_name": "DNPEP Is a Prognostic Marker of Aggressive CLL",
"section_num": "2.2."
},
{
"section_content": "Further analysis was directed towards validating the prognostic power of the identified genes by analyzing the time to treatment and overall survival responses using an independent transcriptomic dataset of 107 CLL patients [17].For both analyses, the hazard ratio associated with ZAP70 mRNA expression was used as a baseline for comparison.Regarding time-to-treatment, a high ZAP70 mRNA expression was associated with a hazard ratio of 1.45 (of note, the clinically used Zap70 expression measure, the percentage of Zap70 protein-expressing cells, was not recorded in the dataset; thus, we used the mRNA expression values).As a measure of their prognostic potential, the HR values associated with the time to treatment for the 32 genes were determined individually (Figure 2A), as well as together (Figure 2B).When analyzed together, the 32-gene set could clearly segregate low and high-risk groups with an HR value of 24.49 (Figure 2B).When the 32 genes were analyzed individually, 8 out of the 32 genes (DNPEP, ARPC4, SMARCA4, TUBA1C, PSMD13, VPS39, EBI3, and ESYT1) were associated with an HR higher that of ZAP70 (Figure 2A), and combined, these eight genes could segregate the patients into high and low-risk groups with an HR of 2.99 (Figure 2C).Additionally, the contribution of each gene to the combined HR was assessed by the iterative removal of each gene, one at a time, from the 32-gene pool (Figure S1A).The strongest eight contributors to the HR were PCSK7, CYC1, VPS72, PSMD13, RPL38, SLC25A11, DNPEP, and ISOC2.This eight-gene signature was associated with an HR value for a time to treatment of 3.47 (Figure S1C). Cancers 2020, 12, 1876 5 of 15 ZAP70 mRNA expression was used as a baseline for comparison.Regarding time-to-treatment, a high ZAP70 mRNA expression was associated with a hazard ratio of 1.45 (of note, the clinically used Zap70 expression measure, the percentage of Zap70 protein-expressing cells, was not recorded in the dataset; thus, we used the mRNA expression values).As a measure of their prognostic potential, the HR values associated with the time to treatment for the 32 genes were determined individually (Figure 2A), as well as together (Figure 2B).When analyzed together, the 32-gene set could clearly segregate low and high-risk groups with an HR value of 24.49 (Figure 2B).When the 32 genes were analyzed individually, 8 out of the 32 genes (DNPEP, ARPC4, SMARCA4, TUBA1C, PSMD13, VPS39, EBI3, and ESYT1) were associated with an HR higher that of ZAP70 (Figure 2A), and combined, these eight genes could segregate the patients into high and low-risk groups with an HR of 2.99 (Figure 2C).Additionally, the contribution of each gene to the combined HR was assessed by the iterative removal of each gene, one at a time, from the 32-gene pool (Figure S1A).The strongest eight contributors to the HR were PCSK7, CYC1, VPS72, PSMD13, RPL38, SLC25A11, DNPEP, and ISOC2.This eight-gene signature was associated with an HR value for a time to treatment of 3.47 (Figure S1C).The same analysis as above was carried out for the overall survival.The reference ZAP70 expression was associated with an HR of 1.94, while the hazard ratio associated with the combination of the 32 BCR kinase-correlating genes was 32.93 (Figure 3B).An analysis of the 32 genes individually The same analysis as above was carried out for the overall survival.The reference ZAP70 expression was associated with an HR of 1.94, while the hazard ratio associated with the combination of the 32 BCR kinase-correlating genes was 32.93 (Figure 3B).An analysis of the 32 genes individually (Figure 3A) revealed eight genes associated with a high HR, and the combination of these eight genes could segregate the patients into high and low-risk groups, with an HR of 6.87 (Figure 3C).This cluster of eight genes consisted of PSMD13, DNPEP, TUBA1C, RPL38, ISOC2, ESYT1, SMARCA4, and ST6GAL1.The contribution of each gene to the combined HR was also assessed by the iterative removal of each gene, one at a time, from the 32-gene pool (Figure S1B).The strongest eight contributors of this analysis included ARPC4, EBI3, RPP7A, PCSK7, DNPEP, Corf61, PDXK, and CWF19L1, and, combined, these eight genes could segregate the CLL patients into high and low-risk groups with an HR of 7.47 (Figure S1D). Cancers 2020, 12, 1876 6 of 15 (Figure 3A) revealed eight genes associated with a high HR, and the combination of these eight genes could segregate the patients into high and low-risk groups, with an HR of 6.87 (Figure 3C).This cluster of eight genes consisted of PSMD13, DNPEP, TUBA1C, RPL38, ISOC2, ESYT1, SMARCA4, and ST6GAL1.The contribution of each gene to the combined HR was also assessed by the iterative removal of each gene, one at a time, from the 32-gene pool (Figure S1B).The strongest eight contributors of this analysis included ARPC4, EBI3, RPP7A, PCSK7, DNPEP, Corf61, PDXK, and CWF19L1, and, combined, these eight genes could segregate the CLL patients into high and low-risk groups with an HR of 7.47 (Figure S1D).Overall, from the analyses at the individual gene level, four genes were found to correlate with both the time-to-treatment and overall survival: DNPEP, PSMD13, ESYT1, and SMARCA4.From the reciprocal analysis (iteratively removing one gene out of the pool of 32), only DNPEP was common between the time-to-treatment and overall survival, and DNPEP emerged as the common gene across the four analyses. ",
"section_name": "DNPEP Is a Prognostic Marker of Aggressive CLL",
"section_num": "2.2."
},
{
"section_content": "By dichotomizing the patients from the Herold dataset into high-and low-DNPEP expressing groups based on the median DNPEP expression, patients with high DNPEP expressions tended to Overall, from the analyses at the individual gene level, four genes were found to correlate with both the time-to-treatment and overall survival: DNPEP, PSMD13, ESYT1, and SMARCA4.From the reciprocal analysis (iteratively removing one gene out of the pool of 32), only DNPEP was common between the time-to-treatment and overall survival, and DNPEP emerged as the common gene across the four analyses. ",
"section_name": "DNPEP as a Prognostic Marker for CLL",
"section_num": "2.3."
},
{
"section_content": "By dichotomizing the patients from the Herold dataset into high-and low-DNPEP expressing groups based on the median DNPEP expression, patients with high DNPEP expressions tended to have a shorter time to treatment, as well as overall survival probability (Figure 4A,B).To further validate DNPEP as a marker for prognosis, DNPEP transcript levels were measured in 34 primary CLL patient samples with real-time quantitative RT-PCR, and a correlation with Rai and Binet staging was determined.The clinical information of the samples is summarized in Table S3.The DNPEP mRNA expression correlated with Rai staging, with the DNPEP expression of the asymptomatic stage (0) being significantly lower than a high risk (III-IV) (p = 0.0019).Similarly, DNPEP transcript levels of medium-risk (I-II) and high-risk (III-IV) groups were also significantly different (p = 0.0014, Figure 4C).The DNPEP expression also correlated with Binet stages, with a high DNPEP expression detected in the intermediate and high-risk groups (B and C stages, p = 0.024, Figure 4D).S3.The DNPEP mRNA expression correlated with Rai staging, with the DNPEP expression of the asymptomatic stage (0) being significantly lower than a high risk (III-IV) (p = 0.0019).Similarly, DNPEP transcript levels of medium-risk (I-II) and high-risk (III-IV) groups were also significantly different (p = 0.0014, Figure 4C).The DNPEP expression also correlated with Binet stages, with a high DNPEP expression detected in the intermediate and high-risk groups (B and C stages, p = 0.024, Figure 4D).To determine whether DNPEP is an independent prognostic marker, it was compared to the immunoglobulin variable heavy chain (IGVH) gene mutational status and 17p13 deletion in a multivariate (Cox) analysis looking at the time to treatment and overall survival using the Herold dataset [17].In the database, 48% of the patients had mutated IGVH, 45% had unmutated IGVH, and 7% was unknown, while 8% of the patients carried 17p13 deletion.A high DNPEP expression (based on the median expression) was a significant prognostic factor in the univariate analysis, both for the To determine whether DNPEP is an independent prognostic marker, it was compared to the immunoglobulin variable heavy chain (IGVH) gene mutational status and 17p13 deletion in a multivariate (Cox) analysis looking at the time to treatment and overall survival using the Herold dataset [17].In the database, 48% of the patients had mutated IGVH, 45% had unmutated IGVH, and 7% was unknown, while 8% of the patients carried 17p13 deletion.A high DNPEP expression (based on the median expression) was a significant prognostic factor in the univariate analysis, both for the time to treatment and overall survival (p = 0.009 and p = 0.01, respectively).In the multivariate Cox regression analysis, a high DNPEP expression remained prognostic for the time to treatment after adjusting for the IGVH mutational status and 17p13 deletion but not for the overall survival (p = 0.032 and p = 0.224, respectively). ",
"section_name": "DNPEP as a Prognostic Marker for CLL",
"section_num": "2.3."
},
{
"section_content": "In order to investigate whether DNPEP is only a biomarker or a possible therapeutic target, a recently developed inhibitor against DNPEP was used [18,19].The DNPEP gene codes for an N-terminal peptidase selective for aspartic acids.The inhibitor, DI93293, used in our study could target two aminopeptidases, DNPEP and the closely related ENPEP (glutamine aminopeptidase).The inhibitor inhibited the peptidase activity of recombinant DNPEP by 95.0% ± 1.4% [18] and by 88% in Mec-1 CLL cell lysate (at 12.5 µM and 25 µM, respectively, Figure S2).The IC 50 value of the inhibitor was 1.4 µM [18]. To test the effect of the DNPEP inhibition on CLL cell survival and drug resistance, Mec-1 CLL cells were cultured alone or on a mesenchymal stromal cell layer expressing the CD40 ligand (CD40L) as a model of the CLL microenvironment.After 24 h of culture, the cells were exposed to a dosage of DI93293 alone or with the Bcl-2/Bcl-X L inhibitor, ABT737, for 48 h, and the induction of cell deaths in Mec-1 cells were determined with annexin V staining (after the exclusion of stromal cells based on their green fluorescent labels).The inhibition of DNPEP alone did not induce any cell deaths but had a modest potentiating effect on ABT737-induced cytotoxicity shown by the CI indices of lower than 1 for all drug dose combinations (calculated by the Chou-Talalay method; Figure 5A, tables under the plots).Stromal support of the CLL cells mostly affected the ABT737 sensitivity, but the potentiating effect of DI93293 was retained (Figure 5B). The effect of DNPEP inhibition on the drug sensitivity of primary, patient-derived CLL cells was also tested using ABT737 and two kinase inhibitors, the PI3K inhibitor, idelalisib, and the BTK inhibitor, ibrutinib (Figure 5C-E).Mononuclear cells isolated from five patients were cultured with bone marrow mesenchymal stromal cell support for 24 h before exposing the cultures to DI93293 alone or in combination with the above drugs for 48 h.The induction of cell death was determined with To-Pro3 staining in the CLL cell population.We found that the inhibition of DNPEP potentiated cell deaths induced by both idelalisib and ibrutinib but not ABT737 (Figure 5D,E). ",
"section_name": "Inhibition of DNPEP Increases Sensitivity to PI3K-and BTK-Inhibitions",
"section_num": "2.4."
},
{
"section_content": "The identification of biomarkers able to indicate the activity of several BcR-linked kinases would improve patient stratification.By using a bi-weight gene expression correlation analysis, we identified genes whose expressions correlated with several BCR-signaling kinases.Genes whose expression could predict patient prognosis correlated with MAPK1 and/or MAPK3, PI3KCD, and ",
"section_name": "Discussion",
"section_num": "3."
},
{
"section_content": "The identification of biomarkers able to indicate the activity of several BcR-linked kinases would improve patient stratification.By using a bi-weight gene expression correlation analysis, we identified genes whose expressions correlated with several BCR-signaling kinases.Genes whose expression could predict patient prognosis correlated with MAPK1 and/or MAPK3, PI3KCD, and ZAP70 and PI3KCD, indicating that the gene expressional program's driving expressions of BcR-signaling kinases are diverse and only partially overlapping. By selecting the genes whose expressions correlated with ZAP70 plus two other BCR-signaling kinase gene, we identified a 32-gene panel.The 32-gene panel could prognose the time to treatment and overall survival probability with very high HR values of 24.49 and 32.93, respectively.The reduction of the number of genes by selecting the top quartile from each analysis, while still producing a prognostic gene set, was nearly not as robust as the 32 genes together, highlighting again that the BCR-induced kinase signaling pathways are only partially overlapping.One gene, aspartic aminopeptidase (DNPEP), showed a high HR value and consistently appeared in all analyses.This biomarker can predict and stratify the risk when CLL patients develop the progressive disease, requiring therapy.An analysis of the DNPEP expression in combination with the IGV H mutation status could be used as a prognostic tool to detect when CLL patients develop the progressive disease and, therefore, when treatment would be required. DNPEP, a member of the M18 peptidase family, is a zinc/manganese-containing metallopeptidase expressed in the cytosol [13].DNPEP cleaves N-terminal aspartate residues from proteins and peptides.Aminopeptidases execute the final step of intracellular protein degradation by trimming or fully degrading peptides produced by the ubiquitin-proteasome pathway.Partial peptide degradation by aminopeptidases may be used to generate MHC-presented peptide antigens, or, after full hydrolysis, the generated free amino acids can be reutilized by the CLL cells for protein synthesis. Although, to date, little is known about the physiological functions of DNPEP, the enhanced expressions of other aminopeptidases in cancer are long known.For example, pancreatic cancer, lymphoma, and leukemia patients have increased leucine aminopeptidase (LAP) activity.Aminopeptidase N (APN) is another type of aminopeptidase implicated in human cancers, such as thyroid cancer [20], ovarian carcinoma [21], breast [22,23], and colon cancer [24].Moreover, APN expression was shown to correlate with poor survival, as well as decreased disease-free survival in colon cancer [24]. Further indicating the potential role of aminopeptidases in cancer, the aminopeptidase inhibitor [25] bestatin showed efficacy in lung cancer [26].New aminopeptidase inhibitors are also emerging, with the best-known example of the prodrug, tosedostat, which is currently in phase II clinical trials for acute myeloid leukemia [25,27]. These results show that aminopeptidases are not only biomarkers of various cancers, but they have a biological functionality in cancer.The free amino acids produced by aminopeptidases support protein synthesis, which some cancers strongly rely on.Accordingly, the inhibition of aminopeptidases by CHR79888 (the active metabolite of tosedostat) induced a typical amino acid deprivation response (AADR) in HL-60 AML cells, including the inhibition of mTOR phosphorylation, reducing the protein synthesis and accumulation of intracellular peptides [27].New emerging results, which show that leukemic cells are sensitive to amino acid deprivation, further support the rationale of aminopeptidase inhibitors as cancer therapeutics [28]. ",
"section_name": "Discussion",
"section_num": "3."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "4."
},
{
"section_content": "B cell transcriptomic datasets were obtained from two main public databases, the ArrayExpress at the EBI (European Bioinformatics Institute, https://www.ebi.ac.uk/arrayexpress/) and the Gene Expression Omnibus (GEO) at NCBI (National Centre for Biotechnology Information, https: //www.ncbi.nlm.nih.gov/geo/).Fourteen B cell transcriptomic datasets were selected.Preprocessing of gene expression datasets included normalization and annotation of the datasets using the open-source programming language \"R\".Datasets from single-channel microarray datasets were normalized using the Robust Multi-Array Average (RMA) method, while two-channel microarray datasets were normalized using the locally weighted scatterplot smoothing (LOESS) method.These normalized datasets were annotated by using BiomaRt and Bioconductor packages. ",
"section_name": "B Cell Transcriptomic Datasets and Preprocessing",
"section_num": "4.1."
},
{
"section_content": "For calculating correlation values between BCR-signaling kinase genes and genes in microarray datasets, the WGCNA package [29] was used, which implements the necessary functions for correlation calculation, gene selection, cluster identification, and network construction.Bi-weight mid-correlation was used to identify genes whose expression correlates with BCR-signaling kinases.The gene list obtained was filtered by applying an absolute threshold and ranking the genes in descending order by the number of datasets the gene was present in for each kinase. ",
"section_name": "Weighted Gene Co-Expression Network Analysis",
"section_num": "4.2."
},
{
"section_content": "For time-to-treatment and overall survival analyses, an independent transcriptomic dataset from 107 CLL samples analyzed on Affymetrix HG-U133 Plus 2.0 chips was used [17,30].For analyses where the combined effect of several genes was assessed, the sample set was divided into two equal-sized risk groups by ranking and splitting the samples at the median according to their estimated prognostic index (risk score).The prognostic index was calculated based on the beta coefficients multiplied by gene expression values using Cox proportional hazard regression: (h i (t)/h 0 (t) = exp(β 1 x 1 + β 2 x 2 + . . .β i x i ), h i is the hazard of the i-th individual, h 0 is the baseline hazard function, exp is the exponent function, x i is the expression value and the β i can obtained from the Cox fitting and the term within the exp function is the prognostic index or risk score.In case a single gene was analyzed, the dataset was divided into two groups based on the median expression level of the gene of interest.Cox proportional hazards models to model the probability of survival and the time to treatment were used where the DNPEP expression (as a binary variable indicating low and high-risk individuals based on the median DNPEP expression) was the only predictor.For multivariate analysis, Cox proportional hazards models were used to model the probability of the survival and treatment with three predictors: IGV H mutational status, p17 mutational status, and DNPEP expression.The Coxph function in the Survival package in R was used to compute the Cox Proportional Hazard regression models and obtain the hazard ratios.The dependent variable, which is a survival object in this linear model, was created using the Surv function in Survival package.Additionally, the Survminer package in R was used to plot the survival curves.The dynamic nomogram of these models, along with the underlying model summaries, have been provided by the DynNom package [31] in R: https://adibmakrooni.shinyapps.io/cxmod10/,https://adibmakrooni.shinyapps.io/cxmod4/,https://adibmakrooni.shinyapps.io/cxmod1/, and https: //adibmakrooni.shinyapps.io/cxmod2/.The expression values of DNPEP in the primary CLL samples were analyzed in the patients grouped by the Rai and Binet clinical CLL stages.Regarding the Rai stages, stage 0 patients (n = 20) were handled as low risk, stage I-II patients (n = 10) as intermediate, and stage III-IV patients (n = 4) as high risk.With Binet stages, only stage A patients (n = 29) and stage B and C patients (n = 5) were distinguished.To compare the mean expression levels of the groups, a Welch two-sample t-test was used in the R statistical environment. ",
"section_name": "Statistical Analyses",
"section_num": "4.3."
},
{
"section_content": "RNA isolated from peripheral blood mononuclear cells of 34 CLL patients were used for gene expression analyses.All patients consented, and the study had ethical approval, research ethics committee: NUI Galway, permission date: 01-06-2015 (duration: 60 months); reference number: CA01355.A summary of the patient clinical data is provided in Table S3. ",
"section_name": "Primary CLL RNA Samples",
"section_num": "4.4."
},
{
"section_content": "Reverse transcription of patient RNA samples was performed using 1 µg of RNA sample with Superscript II (Invitrogen, Waltham, MA, USA), according to the manufacturer's instructions.The expression of genes was analyzed by using PrimeTime Predesigned qPCR assays (Integrated DNA Technologies, IDT, Coralville, IA, USA).Abelson murine leukemia viral oncogene homolog 1 (ABL1) was used as a housekeeping gene for normalization.The TaqMan primer and probe sets for all genes (ABL1, ZAP70, and DNPEP) were predesigned sets purchased from Integrated DNA Technology.For the quantitative polymerase chain reaction (qPCR), Agilent Brilliant III qPCR master mix was used.Each qPCR was performed in a 6-µL reaction volume containing 0.3 µL of cDNA, 2.1-µL H 2 O, 3.0 µL of qPCR master mix, and 0.6 µL of the qPCR primer assay.Initial denaturation at 95 • C for 15 min was followed by 40 cycles of a denaturation step at 95 • C for 15 s, an annealing step at 57.5 • C for 30 s, and an extension step at 72 • C for 30 s on a Roche LC480 qPCR light cycler. The threshold cycle (Ct) value was calculated as the cycle number at which the fluorescence of the reporter reached a fixed threshold.The calculation of the relative expression of the target genes in comparison to the reference gene was performed in an R statistical environment (Supplementary R-Script). ",
"section_name": "Reverse Transcription-Coupled Quantitative Real-Time Polymerase Chain Reaction",
"section_num": "4.5."
},
{
"section_content": "The Mec-1 CLL cell line was cultured in RPMI-1640 medium (Sigma, St. Louis, MO, USA) containing 10% heat-inactivated FBS (Hyclone, Marlborough, MA, USA), penicillin (100 U/mL), streptomycin (100 µg/mL) (Sigma), and L-glutamine (2 mM).The bone marrow mesenchymal stromal cell line, HS-5, was cultured in DMEM (high-glucose, Gibco, Waltham, MA, USA) supplemented with 10% FBS (Hyclone), GlutaMAX-I (2 mM) (Gibco), penicillin (100 U/mL), streptomycin (100 µg/mL), and 1-mM sodium pyruvate (Sigma).Primary CLL samples were generated from peripheral blood samples by isolating the mononuclear cell (MNC) fraction with Ficoll gradient-centrifugation [32].MNCs were stored in liquid nitrogen until use.Upon revival of the cells, viability was determined with trypan blue staining.Only samples with viability above 60% were used.All patients consented according to the local Ethical Committee Regulations.Primary CLL cells were grown on an HS-5 stromal feeder layer in RPMI-1640 medium (Sigma) containing 10% FBS, penicillin (100 U/mL), streptomycin (100 µg/mL), and L-glutamine (2 mM).The HS-5 cells were stably transfected with GFP (green fluorescent protein) to enable their identification and separation from the CLL cell population in downstream analyses. ",
"section_name": "Cell Culture",
"section_num": "4.6."
},
{
"section_content": "Mec-1 cell viability was quantified with annexin V staining.Cells were collected and stained with annexin V-APC (Immunotools) in annexin V buffer (10-mM HEPES/NaOH, pH 7.5, 140-mM NaCl, and 2.5-mM CaCl 2 ) for 15 min on ice in the dark.Samples were analyzed on a FACS Canto II flow cytometer.The viability of primary CLL cells was determined using the viability dye, To-Pro-3 (Molecular Probes), according to the manufacturer's protocols.The samples were analyzed using the BD FACS Canto II flow cytometer (BD Bioscience, San Diego, CA, USA) by collecting 30,000 events.HS-5 cells were excluded from the analysis by gating out GFP + /FSC high events.Statistical analysis was performed using FCSExpress (DeNovo Software Inc., Pasadena CA, USA) and GraphPad Prism (GraphPad Software Inc., La Jolla, San Diego, CA, USA) software packages. ",
"section_name": "Measurement of Cell Viability",
"section_num": "4.7."
},
{
"section_content": "L-aspartic acid 7-amido-4-methylcoumarin (Asp-AMC) was used as the substrate to analyze the DNPEP enzyme activity.Mec-1 cell lysate, as the source of DNPEP enzyme, was obtained by lysing 1 × 10 6 Mec-1 cells in 100 uL of cell lysis buffer (250-µM HEPES, pH 7.4, 25-µM CHAPS, and 2-µM DTT).Ten microliters of cell lysate was dispensed into a black, clear-bottom 96-well plate and incubated with 100-µL assay buffer (50-mM Tris-HCl, pH 7.5) containing 22-µM ASP-AMC substrate at 37 • C; As a negative control, DNPEP activity was blocked by adding 0.5-mM ZnCl 2 to the reaction.DNPEP activity was monitored kinetically by determining the fluorescence produced in every 60 s over 30 min.Enzyme activity was calculated from the linear portion of the fluorescent intensity graphs.The effect of the DNPEP inhibitor (DI93293) was calculated according to the following formula: activity score = ((RFI DI -RFI u )/(RFI u -RFI Zn )) × 100%, where RFI u : enzyme activity of untreated sample, RFI DI : enzyme activity of DI-treated sample, and RFI Zn : same of sample containing ZnCl 2 . ",
"section_name": "DNPEP Enzyme Activity Assay",
"section_num": "4.8."
},
{
"section_content": "In conclusion, by using gene co-expression analysis, we identified DNPEP as a biomarker to identify CLL patients with poor prognosis.This biomarker can predict and stratify the risk of when CLL patients develop the progressive disease, requiring therapy.This is comparable to the Rai and Binet clinical CLL staging systems.DNPEP, in combination with IgHV, could be used as a prognostic tool for when CLL patients develop the progressive disease, requiring therapy.Furthermore, our results warrant further study evaluations of DNPEP as a novel target for combination therapy to reverse drug resistance. ",
"section_name": "Conclusions",
"section_num": "5."
}
] |
[
{
"section_content": "The DNPEP inhibitor (DI93293) was a kind gift from Philip Kiser, currently affiliated with the University of California Irvine.The independent dataset used, taken from 107 CLL patients, was collected by Tobias Herold (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE22762) et al. [17]. ",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "Funding: This research was funded by the Irish Cancer Society and Science Foundation Ireland (BCNI, 14/ICS/B3042). ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The following are available online at http://www.mdpi.com/2072-6694/12/7/1876/s1. Figure S1: Effect of BCR-kinase correlating genes in time to treatment and overall survival probability of CLL patients, Figure S2: DNPEP inhibition by DI93293 kinetic assay, Table S1: Databases and selected samples for co-expression analysis, Table S2: List of genes correlating with ZAP-70 and at least two other BCR-signaling kinase genes, Table S3 The authors declare no conflict of interest. ",
"section_name": "Supplementary Materials:",
"section_num": null
},
{
"section_content": "The following are available online at http://www.mdpi.com/2072-6694/12/7/1876/s1. Figure S1: Effect of BCR-kinase correlating genes in time to treatment and overall survival probability of CLL patients, Figure S2: DNPEP inhibition by DI93293 kinetic assay, Table S1: Databases and selected samples for co-expression analysis, Table S2: List of genes correlating with ZAP-70 and at least two other BCR-signaling kinase genes, Table S3 ",
"section_name": "Supplementary Materials:",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.1186/1479-5876-12-207
|
Targeted DNA vaccines eliciting crossreactive anti-idiotypic antibody responses against human B cell malignancies in mice
|
Therapeutic idiotypic (Id) vaccination is an experimental treatment for selected B cell malignancies. A broader use of Id-based vaccination, however, is hampered by the complexity and costs due to the individualized production of protein vaccines. These limitations may be overcome by targeted DNA vaccines encoding stereotyped immunoglobulin V regions of B cell malignancies. We have here investigated whether such vaccines might elicit cross-reactive immune responses thus offering the possibility to immunize subsets of patients with the same vaccine.Fusion vaccines targeting patient Id to mouse Major Histocompatibility Complex (MHC) class II molecules (chimeric mouse/human) or chemokine receptors (fully human) on antigen-presenting cells (APC) were genetically constructed for two Chronic Lymphocytic Leukemia (CLL) patients and one prototypic stereotyped B-cell receptor (BCR) commonly expressed by Hepatitis C Virus (HCV)-associated Non Hodgkin Lymphoma (NHL). The A20 murine B lymphoma cells were engineered to express prototypic HCV-associated B cell lymphoma BCR. Anti-Id antibody responses were studied against stereotyped and non-stereotyped BCRs on CLL patients' cells as well as transfected A20 cells.DNA vaccination of mice with Id vaccines that target APC elicited increased amounts of antibodies specific for the patient's Id as compared with non targeted control vaccines. Anti-Id antibodies cross-reacted between CLL cells with closely related BCR. A20 cells engineered to express patients' V regions were not tumorigenic in mice, preventing tumor challenge experiments.These findings provide experimental support for use of APC-targeted fusion Id DNA vaccines for the treatment of B cell lymphoma and CLL that express stereotyped BCRs.
|
[
{
"section_content": "B cell malignancies express a highly tumor-specific antigen, the variable (V) regions of the monoclonal immunoglobulin (Ig), which contain antigenic determinants called idiotopes collectively known as idiotype (Id).Protein Id vaccination has been pursued as a therapeutic approach to B cell malignancies over the last 20 years [1].Immunologic and clinical responses have been detected [1], whereas demonstration of clinical benefit is so far limited to follicular lymphoma [2,3]. Because of the very nature of the antigen (Ag) (i.e., Id), large scale clinical application of protein Id vaccination is limited by the need to prepare a custom-made vaccine for each and every patient.Overcoming this problem, DNA vaccination holds promise to streamline tailor-made vaccine manufacture by circumventing the need for purification of Ig protein (or derivatives thereof), conjugation to carrier protein (e.g.KLH) and administration of adjuvants.However, although effective in rodents, DNA vaccination has met with limited success thus far in humans due to low potency of vaccines [4].The poor immunogenicity of DNA vaccination can be improved by several means such as improved vector design and efficient electroporation [5].Another strategy is based on the finding that targeting of Ag to antigen-presenting cells (APC) enhances immunogenicity, as shown for chemical antibody (Ab)-Ag conjugates [6,7] and Ab-Ag fusion proteins [8,9].Thus, we [10] and others [11,12] cloned DNA constructs encoding proteins that target Ag to APC.When such constructs were injected s.c. or i.m., combined with electroporation, transfected host cells secreted fusion proteins that targeted APC for enhanced immune responses [10].In our previous studies, we used homodimeric Ig-based vaccines (Vaccibodies, VB), each chain consisting of a targeting unit, a dimerization unit and an antigenic unit.The dimerization unit consists of a shortened hinge region from hIgG3 whereas the N-terminal targeting unit can consist of either single chain fragment variable (scFv) specific for surface molecules on APC such as mouse MHC class II [10], mouse CD40 [13], human TLR2 and CD14 [14], or natural ligands such as the mouse chemokines CCL3 (mCCL3) and CCL5 (mCCL5) [15] and human CCL3 [16].Depending on targeting strategy, such VB proteins had a 10-10,000 fold increased efficiency to stimulate CD4 + T cells in vitro in mice [10,13,15,16] and humans [14,16].Moreover, DNA Vaccibodies elicited superior antibody and T cell responses in mice, as well as greatly enhanced tumor protection [10,13,15,16].In a stepwise, translational endeavour, the first fully murine Vaccibodies [10] have been extended to chimeric murine/human Vaccibodies, including tailormade Vaccibodies for multiple myeloma patients [17]. A complementary strategy to streamline clinical Id vaccination is to exploit the high similarity of Ig V regions expressed by molecularly identified subgroups of patients with B cell malignancies.For example, the molecular characterization of Hepatitis C Virus (HCV) related lymphomas showed that more than 70% of these cases expressed either IGKV3-20 or IGKV3-15 light chains [18][19][20], with a high degree of homology between individual lymphomas.Moreover, IGHV1-69 is expressed as the partner of IGKV3-20 or IGKV3-15 in up to 70% of HCV-related lymphomas [18,20].Such commonly expressed B cell receptors (BCR) are called stereotyped receptors.Stereotyped BCRs are found also in several non HCV-associated B cell malignancies, such as MALT lymphomas [21][22][23] and Chronic Lymphocytic Leukemia (CLL) [24][25][26].The analysis of VH CDR3 in more than 7000 VH (IGHV-IGHD-IGHJ) sequences from patients with CLL has established that CLL comprises two distinct categories: one with stereotyped and the other with heterogeneous BCR, in an approximate ratio of 1:2 [27].Thus, it could be envisioned that a number of off-theshelf Id vaccines for molecularly identifiable subgroups of patients could be developed, obviating the need to tailormake Id-vaccines for every patient.Although it is not known whether these Ids are immunogenic in the majority of patients, such off-the-shelf Id vaccines could cover up to 30% of patients with selected B cell malignancies, thus affording substantial savings in time and costs associated with Id vaccine manufacture. On these premises, we have here produced fully human chemokine-Id fusion DNA Vaccibodies which due to cross-species reactivity of chemokines could be tested as DNA vaccines in mice.Moreover, using a panel of CLL patients' cells and a mouse model for HCV-associated B cell lymphomas we explored the possibility of inducing cross-reactive anti-Id antibody responses following immunization with VB expressing a stereotyped B cell receptor. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Patients diagnosed with CLL (see Table 1) were seen at the Department of Haematology outpatient clinic, Oslo University Hospital, Rikshospitalet, Oslo, Norway.Blood samples from 5 patients were procured following written informed consent using protocols approved by the Regional Committee for Medical and Research Ethics, South-East Norway.Blood samples were procured in tubes containing ACD as anticoagulant.Experiments were conducted on purified mononuclear blood cells. ",
"section_name": "Patient material",
"section_num": null
},
{
"section_content": "Cells were stained with primary reagents and appropriate secondary reagents or control as indicated.The following biotinylated mAbs were used: anti human IgG (HP6017, Zymed), anti mouse IgD (TIB149, ATCC), anti mouse Ck (clone 187.1), anti mouse IgG1 a (clone 10.9, BD Pharmingen), anti mouse IgG2a a (clone 8.3, BD Pharmingen), anti mouse IgG2a b (clone 5.7, BD Pharmingen).Quantification of surface antigen on CLL cells was performed using mouse mAbs targeting human λ (clone 4C2) and human κ L chains (clone A8B5), and human IgM (clone 1030) from Diatec, Oslo, Norway, and the bead based Cellquant Calibrator kit (BioCytex, Marseille, France) according to the manufacturer's guidelines [28].Cells (20,000) were acquired on a FacsCalibur (BD).Flow cytometry files were analyzed on CellQuest (BD) and Weasel v3.0 (http://www.wehi.edu.au). ",
"section_name": "Flow cytometry",
"section_num": null
},
{
"section_content": "BALB/c mice were obtained from Taconic (Ry, Denmark).B10.D2.C-TCRα a /Bo (H-2 d , Ig haplotype IgH-C b ) mice were bred in house.These congenic mice are identical to B10.D2 except being congenic for the TCRα a region [29].The studies were approved by the National Committee for Animal Experiments (Oslo, Norway).HEK 293E cells were from ATCC.The murine lymphoma Esb/MP cells [30] were kindly provided by Jo Van Damme (Leuven, Belgium).The human Burkitt's lymphomas DG-75 [31] and PA682 [32] were obtained from ATCC and kindly provided by Keith Thompson (Oslo, Norway), respectively. Identification of V H and V L genes from CLL patients and assembly of scFv V H and V L tumor-specific genes were cloned from preparations of cDNA from PBMCs of each patient.Tumorspecific transcripts were identified by PCR as previously described [33].Sequence data were analyzed using the IMGT database (http://www.imgt.org)and the IMGT/V-QUEST tool [34].Nucleotide sequences were aligned using BLAST (http:// blast.ncbi.nlm.nih.gov/).Amino acid sequences were aligned using clustal omega (http://www.ebi.ac.uk/Tools/msa/clus talo/) and GeneDoc (http://nrbsc.org/gfx/genedoc/) for analysis of conservative, semi-conservative and non conservative changes.Tumor-specific V H and V L genes from CLL patients were arranged in a single chain variable region fragment (scFv) in VH-VL orientation by PCR Soeing (Synthesis by Overlap Extension) essentially as described previously [33]. ",
"section_name": "Mice and cell lines",
"section_num": null
},
{
"section_content": "Plasmids encoding V (D) J H (VH1-69, VD3-22, VJ4) and VJ κ (VK3-20, VJ1) sequences from patients 1 and 2, respectively, were kindly provided by V. De Re (Aviano, Italy) and assembled into scFv as described above.This association has been shown to be representative of stereotyped Id molecules expressed by up to 70% of B cell malignancies associated with HCV chronic infection [18,20]. ",
"section_name": "Assembly of stereotyped scFv for Hepatitis C virus-associated B cell lymphomas",
"section_num": null
},
{
"section_content": "The resulting scFv from individual CLL patients or the prototypic stereotyped BCR from HCV-associated B lymphoma were cloned C-terminal as antigenic units into the previously described VB format [10].In Vaccibodies used in this study, the N-terminal targeting unit consisted either of a mouse scFv from the 14-4-4S mAb specific for I-E mouse MHC class II molecules [10], or the human chemokine LD78β (CCL3-L1) which binds CCR1, CCR3 and CCR5 [16].Control non-targeted Vaccibodies encoded a mouse scFv specific for the hapten NIP (5-iodo-4-hydroxy-3-nitrophenylacetyl), an antigen which is not found in the mouse tissue [10].The dimerization unit consisted of a shortened hinge (h1 + h4) and C H 3 of human IgG3 [10]. ",
"section_name": "Assembly of patient-specific and cross-reactive Vaccibodies",
"section_num": null
},
{
"section_content": "Expression and function of chimeric VB constructs was determined on supernatants from transiently transfected HEK 293E cells.To comparatively measure concentration of Vaccibodies expressing different targeting and antigenic units, an ELISA detecting the presence of human IgG3C H 3 in the dimerization unit was set up: mAb MCA878G (binds human IgG3C H 3, AB Serotec) as coat and biotinylated mAb HP6017 (binds to a different epitope in human IgG3C H 3) for detection.Binding to MHC class II was verified by admixing I-E d -specific VBcontaining supernatants and BALB/c (I-E d+ ) A20 B lymphoma cells.Bound VB proteins were detected as previously described [17].NIP-specific Vaccibodies were tested for their ability to bind to NIP-BSA (conjugated in-house) as previously described [17].Chemotactic activity of LD78β (CCL3-L1) Vaccibodies on the mouse Esb-MP T cell lymphoma was tested by a transwell plate (Corning), as previously described [16].The results (mean + SE of duplicate samples) are presented as chemotactic index, defined as the fold increase in the number of migrating cells in the presence of chemotactic factors over the spontaneous cell migration (i.e., in the presence of medium alone). ",
"section_name": "Vaccine protein production and assessment of targeting properties",
"section_num": null
},
{
"section_content": "Heterohybridomas secreting tumor-specific Ig were generated from PBMCs of two patients with CLL by standard procedures [35].Hybridomas were screened by ELISA for expression of an Ig of expected H and L-chain isotypes.H-chain of hybridomas was verified by V H sequencing. Hybridomas with V H sequence fully matching that retrieved from patients' CLL cells were selected for further study. ",
"section_name": "Purification of patient tumor Ig protein",
"section_num": null
},
{
"section_content": "V (D) J H regions from patient 1 (V H P1) and VJ L regions from patient 2 (V κ P2) were cloned into independent vectors that had been developed for membrane expression (but not secretion) of mouse IgDκ [36].In particular, V H P1mIgDpLNOK vector expressing G418 resistance contains downstream of V H P1 the murine germline IgD sequence (IgD a allotype) in which the 3' secretory exons had been eliminated, whereas V κ P2 was cloned in the pMUSmCκ expressing mouse constant κ region and zeocin resistance (Tuva Hereng and Bjarne Bogen, unpublished) to generate V κ P2MUSKAP.A20 BALB/c B lymphoma cells that express an endogenous IgG2aκ and MHC class II (including I-E d ) were transfected with either or both vectors by electroporation, grown in selection medium (G418 and/or zeocin), and cloned by limiting dilution.To screen for A20 transfectants expressing patient V H , cells were stained with anti-IgD TIB149 (ATCC).Transfectants were selected by flow sorting for high IgD expression followed by cell culture and re-sorting.Expression of VH1-69 and VK3-20 was assessed by RT-PCR using the following primers: for VH1-69, forward GTGCAGCTGGTGC AGTCT and reverse TCCCTGGCCCCAATAGAAGT; for VK3-20 forward TTGTGTTGACGCAGTCTCCAG and reverse TTGATTTCCACCTTGGTCCCT. As a negative control, we used an A20 cell that expressed IgDκ with V regions derived from the unrelated syngeneic Ab2-1.4 hybridoma [36]. ",
"section_name": "Generation of mouse B lymphoma cells (A20) that express a stereotyped BCR of HCV-associated B cell lymphomas",
"section_num": null
},
{
"section_content": "VB plasmids were purified with Endofree® Plasmid Mega Kit (Qiagen).25 μL solution of 0.5 mg/mL VB DNA in sterile 0.9% NaCl (total 25 μg per mouse) was injected intradermally in the lower back of mice, on both sides, followed by electroporation using Derma Vax™ (Cyto Pulse Sciences, MD, USA).Groups consisted of 3 to 7 mice. ",
"section_name": "Mouse immunization",
"section_num": null
},
{
"section_content": "Blood samples were obtained at different time-points from the leg vein of vaccinated mice.Sera were tested by ELISA for reactivity against the patients' CLL-derived monoclonal IgM or control isotype-matched IgM, or human IgG3, as coat.Bound antibodies were detected by either biotinylated mAb 187.1 (detects murine Cκ), antimouse IgG1 a (clone 10.9 BD Pharmingen) or anti-mouse IgG2a a (clone 8.3 BD Pharmingen).The endpoint titres were recorded as the final serum dilution giving a signal above a fixed concentration of alkaline phosphataseconjugated goat anti-human IgM (Sigma) or biotinylated mouse anti-human IgG (HP6017). Sera were also used to stain PBMC from different CLL patients.Bound mouse antibodies were detected with biotinylated mAb 187.1 followed by streptavidin PerCP.Surface Ig expression by CLL cells was assessed by Cell-Quant (Biocytex), measuring the average Ig surface number by analyzing 20,000 cells. Sera from immunized BALB/c or B10.D2.C-TCRa a /Bo (H-2 d IgH-C b ) mice were used to stain A20 transfectants expressing human IGHV1-69, or human IGKV3-20, or both.Following blocking with PBS with BSA and heatinactivated rat serum, transfectants were admixed with sera.Bound mouse antibodies were detected with biotinylated anti-mouse IgG1 a for BALB/c sera or antimouse IgG2a b for B10.D2.C-TCRa a sera. ",
"section_name": "Measurement of antibody responses and assessment of specificity of anti-scFv antibodies",
"section_num": null
},
{
"section_content": "BALB/c mice were injected s.c. with 3 × 10 6 parental A20 cells or with A20 cells stably transfected with either VH1-69, or VK3-20 or both, and followed up for tumor growth. ",
"section_name": "Injection of transfected A20 cells in immunocompetent mice",
"section_num": null
},
{
"section_content": "Analysis of variance and regression analysis were conducted on all treatment arms.Results are presented as comparison of slopes of the antibody response in different treatment arms across serum dilutions. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "The characteristics of the patients' CLL cell BCR included in this study are presented in Table 1.The CLLspecific V regions were identified in each case as identical VDJ H and VJ L sequences repeatedly obtained after cloning of PCR products.The corresponding scFv was then assembled for two patients (CLL106 and CLL107) and cloned into the antigenic unit of various VB scaffolds.According to previously published results on other Vaccibodies [10,[13][14][15][16][17], transiently transfected HEK 293 cells were expected to secrete homodimeric fusion proteins consisting of i) two targeting units: either mouse scFv specific for mouse MHC class II molecules (I-E d ), or the hapten NIP (non targeted negative control), or two human CCL3 (LD78β isoform) chemokine moieties, ii) two dimerization units: human hinge-CH3 held together by disulfide bonds and non-covalent interactions, and iii) two antigenic units: either human scFv of CLL origin (patients CLL106 and CLL107) or prototypic stereotyped V H and V L associated with HCV-associated B lymphomas (Figure 1A).ELISA on supernatants of transiently transfected 293E cells demonstrated similar levels of secretion of the fusion protein for each construct (Figure 1B and data not shown).The targeting units of the respective bivalent VB proteins retained their functional properties since anti-MHC II VB bound MHC class II + ve A20 cells (Figure 1C) and LD78β VB chemoattracted mouse Esb/MP cells (Figure 1D).Non targeted anti-NIP VB neither bound A20 cells (Figure 1C) nor chemoattracted Esb/MP cells, whereas it bound NIP-BSA in ELISA (data not shown).Taken together, these results of Figure 1 are consistent with previous extensive characterizations of VB molecules expressing other antigenic units [10,[13][14][15][16][17]. ",
"section_name": "Identification and assembly of CLL-derived V genes into Vaccibodies",
"section_num": null
},
{
"section_content": "Sera from mice that had been immunized once with different VB constructs were tested for recognition of the corresponding tumor Ig by ELISA at week 2, 4 and 9 after immunization.To this end, the BCR of patient CLL106 was expressed by rescue hybridoma as a secreted IgM used for coating ELISA plates.Improved antibody responses were observed in all mice immunized with either MHC class-II-or chemokine receptor-targeted VB, as compared with mice immunized with the control non targeted NIP-specific VB (Figure 2A and data not shown).By week 9, the effect of targeting was less striking but nevertheless observed, in keeping with previous findings [10,13,16,17,37].Induction of antibodies was specific for the antigenic unit in the immunogen since sera from mice that had been immunized with VB encoding patient CLL107 scFv Id as antigenic unit did not recognize patient CLL106 IgM in ELISA at any time point (Figure 2A and data not shown).In order to assess the nature of the antibody response induced by the different Vaccibodies, the IgG1 and IgG2a components were compared at different time points.The targeting unit appeared to influence antibody isotype since a trend toward a predominant IgG2a or IgG1 antibodies were observed in sera of mice that had been immunized with chemokine receptor-or MHC class II-targeted VB, respectively, starting from week 4 and peaking at week 9 (Figure 2B and data not shown), in line with previous reports [10,15,37,38]. We next investigated if sera from the targeted VB 106-immunized mice bound BCR on CLL cells.Sera were admixed with PBMCs from patient CLL106 as well as four other CLL patients (Table 1).Sera from all immunized mice had antibodies binding 106 CLL cells in titres >200 (Figure 3A and data not shown).Strikingly, the sera also cross-reacted with CLL cells from CLL103 and to a lesser extent with CLL111 CLL cells, but not with CLL107 and CLL116 CLL cells (Figure 3B).The CLL cells from these two patients (CLL103, CLL111) expressed Vλ and VH genes of the same family as CLL106 (Table 1 and Figure 3).At the protein level, the L chain V-regions of CLL111 and CLL103 CLL cells were close to identical with CLL106 (Figure 3C).Moreover, the VH were of the same family (IGHV3) and CLL cells were identical to CLL106 in 82% (CLL103) and 83% (CLL111) of the VH amino acids (Figure 3D).The results suggested that the elicited antibodies crossreacted to homologous sequences or combinatorial V H / V L determinants.The lower level of staining of CLL111 cells could be explained by IGLV3-21 being diluted by a second productive L chain rearrangement found in these CLL cells (IGKV3-20*01 F/IGKJ1*01 F) expressed by the vast majority of cells (Table 1, Figure 3B and data not shown), reducing the level of V L and V H /V L determinants on these cells.All CLL cells tested expressed low but comparable levels of surface Ig (data not shown).The lack of unspecific staining due to recognition of human IgM by mouse antibodies was ruled out by lack of staining of patient CLL107 and CLL116 cells (Figure 3B and data not shown). ",
"section_name": "Analysis of the antibody responses induced by DNA Vaccibody immunization",
"section_num": null
},
{
"section_content": "A sizeable proportion of B cell lymphoproliferative diseases, particularly HCV-associated B cell lymphomas, express IGHV1-69 and IGKV3-20 [18,20].Thus, we identified prototypic IGHV1-69 and IGKV3-20 genes obtained from two different HCV-associated lymphomas selected as having a representative predicted amino acid sequence.Hence, we constructed a mouse model suitable for the development of an Id vaccine expressing these stereotyped V regions.In order to reduce the possibility that immunocompetent mice spontaneously rejected cells expressing human Ig, prototypic IGHV1-69 and IGKV3-20 V (D) J from patient 1 and 2, respectively, were cloned with mouse δ and κ constant regions, respectively, and transfected into the BALB/c B cell lymphoma A20.It should be noted that the mouse δ construct was engineered so that it was only expressed on the membrane but was not secreted [36].The following three human/mouse Ig transfectants were generated: (i) A20 IGHV1-69, transfected with IGHV1-69 alone, (ii) A20 IGKV3-20 transfected with IGKV3-20 alone, and iii) A20 IGHV1-69/IGKV3-20 transfected with both IGHV1-69 and IGKV3-20 (Figure 4A).In addition to the transfected chains, A20 cells express an endogenous IgG2a,κ and all types of transfected cells retained expression of the endogenous BCR (data not shown).Surface expression of transfected IGHV1-69-mouse Cδ hybrid gene was confirmed by flow cytometry (Figure 4A).Note also that the expression of IgD/IGHV1-69 in the absence of IGKV3-20 (Figure 4A, bottom right) was detected at the same (or higher) levels than in the presence of VK3-20 (Figure 4A, top right).As A20 cells express endogenous κ chains (κ Endo ) IgD/ IGHV1-69 could pair with this κ Endo . The lack of a validated anti-human IGKV3-20 mAb negated flow cytometric analysis of V Κ expression, which was therefore screened by RT-PCR (Figure 4B).Thus, no formal proof of surface expression of the human IGHV1-69/IGKV3-20 pair on transfected A20 cells could be obtained, although this is likely to be the case.Transfectants bona fide expressing human IGHV1-69/IGKV3-20 were sorted three times to further select efficiently transfected cells. ",
"section_name": "Construction of a mouse model for B cell lymphomas expressing a prototypic human HCV-associated stereotyped BCR",
"section_num": null
},
{
"section_content": "Mice were immunized with LD78β-VB having an antigenic unit comprised of prototypic HCV-associated B lymphoma BCR with IGHV1-69 and IGKV3-20 linked in a scFv format.In terms of secondary detection of bound murine antibodies, it is notable that A20 cells endogenously express surface IgG2a, precluding detection of bound serum IgG2a.Immunized mice had low levels of serum IgG1 antibodies that stained IGHV1-69/ IGKV3-20 A20 cells (data not shown).To be able to visualize IgG2a responses, we also immunized a strain of mice that differ in Ig H-chain allotype, permitting use of anti-IgG2a allotype-specific antibodies in flow cytometry.Hence, B10.D2.C-TCRα a (H-2 d , Ig haplotype IgH-C b ) mice (see Materials) were immunized with the same VB as above.Sera from mice that had been immunized once bound A20 IGHV1-69/IGKV3-20 as well as A20 IGHV1-69 cells, but not untransfected A20 or A20 IGKV3-20 cells (Figure 5A).Antibody titres were more than 1:3200 (Figure 5B).Note that as A20 expresses endogenous gamma chains (γ Endo ) as well as κ (κ Endo ), the latter cells could express γ Endo -chains paired with IGKV3-20.Similarly, the IGHV1-69 transfectant could express this heavy chain paired with κ Endo .We further tested sera for binding to DG-75 [31], a sIgMκ + Burkitt's lymphoma cell line that expresses very similar IGKV3-20 (93.8% identity of the Vκ amino acids) but dissimilar VH (IGHV3-23) (only 51.5% identity of VH amino acids) (Figure 5C and data not shown).In spite of the expression of the correct IGKV3-20 L chain, sera failed to stain these cells, suggesting that the elicited antibodies were predominantly specific for V H .However, some anti-Id .Sera from mice that had been DNA vaccinated with the patient CLL106-specific VB targeted by LD78β, were tested in flow cytometry for binding to patient 106 CLL cells.Shaded histogram, serum from mock immunized mouse, black line serum from a representative immunized mouse (n = 5/group).(B) The same sera were tested for cross reactivity against a panel of CLL cells obtained from different patients (see Table 1).Data for a representative mouse serum are shown at 1:200 dilution.(C) & (D) Alignment of V L (C) and V H regions (D) from patients CLL106, CLL103 and CLL111.V regions amino acid sequences were aligned using GeneDoc.Amino acids are color coded according to charge (negative: D, E; positive: H, K, R), or the chemical properties of side chains (i.e.amide: N, Q; alcohol: S, T; aliphatic: L, I, V; aromatic: F, Y, W; small size: A, G; sulfur atom: M, C; or other: P, see key for color code). antibodies most likely bound V L /V H combinatorial determinants since A20 cells expressing VH1-69/VK3-20 stained brighter than A20 cells expressing only VH1-69.This result was obtained with sera from 4 out of 5 mice (Figure 5A, arrows/circles), indicating a contribution of combinatorial V H /V L epitopes.The sera from all mice failed to bind A20 cells expressing an unrelated IgD (Ab2-1.4,see Methods and Figure 5C), as well as a Burkitt's lymphoma control (PA682, see Methods). ",
"section_name": "Analysis of antibodies induced by DNA immunization with Vaccibodies expressing a stereotyped BCR found in HCV-associated B lymphomas",
"section_num": null
},
{
"section_content": "Three BALB/c mice per group were injected s.c. with 3 × 10 6 either parental A20 cells or IGHV1-69/IGKV3-20 transfected A20 cells.Two out of three mice that had been injected with parental A20 cells developed tumors, whereas no mice that had been injected with IGHV1-69/ IGKV3-20 A20 cells developed tumors, presumably due to immunogenicity of the xenogeneic (human) Ig V regions sequences in mice.These results are reminiscent of our previous finding in multiple myeloma, where the tumorigenicity of MOPC315.36 was decreased by stable expression of human V genes [17].The lack of tumorigenicity of the A20 transfectant in immunocompetent mice precluded both prophylactic and therapeutic antitumor vaccination experiments. ",
"section_name": "Growth of transfected A20 cells in BALB/c mice",
"section_num": null
},
{
"section_content": "Herein we describe experiments aimed at generating Id vaccines for therapeutic Id vaccination of groups of patients with B cell malignancies expressing stereotyped BCRs.First, we demonstrate the feasibility of constructing fully human Id Vaccibodies with maintenance of both Id epitopes and functionality of targeting units (i.e., human CCL3 chemokine LD78β that cross-react with mouse CCRs) [16].Similar to fully murine and chimeric mouse/human vaccibodies, targeting antigen delivery to APC by human chemokine resulted in augmented immune responses in mice as compared with non-targeted control DNA Vaccibodies [16].Since these vaccines are fully human and isoform LD78β of human CCL3 bind cells expressing Rhesus macaque CCR5 [16], they are suited for both preclinical immunogenicity and regulatory toxicology studies in view of clinical application.It may be anticipated that targeting scFv Id to APC by LD78β could result in increased anti-Id responses in patients.Furthermore, DNA vaccination combined with electroporation is already employed in clinical trials for melanoma and prostate cancer, with mild to moderate, reversible side effects [39]. Second, we explore a complementary approach to further streamline clinical application of Id vaccine for B cell malignancies. Antibodies elicited in mice by Id DNA Vaccibodies constructed for a CLL patient showed cross-reactivity with CLL cells from some other patients.The most plausible explanation is that a fraction of mouse antibodies recognized epitope(s) displayed by the V L and/or V L /V H of the cross-reactive CLLs, since the CLL cells expressed nearly identical IGLV3-21 and similar IGHV3 family genes.With the HCV-NHL construct, the induced antibodies bound the transfected A20 cells but failed to bind a Burkitt's lymphoma expressing the correct Vκ but not the corresponding VH, indicating a dominant anti-VH response.Even so, a contribution of the Vκ was however detected as VK3-20 + VH1-69 + transfectants stained brighter than Vk Endogenous /VH1-69 only cells (that express an endogenous Vκ), suggesting responses towards VL/VH combinatorial idiotypes.Taken together, it is suggested that immunization of mice with fully human targeted scFv Id DNA vaccines could elicit antibodies that may react with either V L , or V H , or V L + V H , the relative proportions differing from case to case. The above observations suggest the possibility of constructing vaccines covering molecularly identified subgroups of patients with B cell malignancies.This idea is supported by evidence of Id cross-recognition by anti-Id mAb [40] as well as cross-reactive responses observed in clinical trials [41][42][43][44][45].A subgroup-specific, \"off-the-shelf\" Id vaccine should elicit a cross-reactive immune response effective against unrelated B cell tumors expressing V regions of the same families, provided that the pattern of somatic mutations is similar between individual tumors.In this respect, the application of criteria developed for clustering stereotyped BCR as based on HCDR3 sequences only [27] do not fully meet the need for identifying patients amenable to immunization with such vaccines, as immune responses following vaccination may be directed to determinants located elsewhere in the V regions [45][46][47].Hence, similarity across the whole V regions should be evaluated.In principle, staining of lymphoma sections or single cell suspensions obtained from biopsy with serum from mouse that had been immunized with the intended Id vaccine could be able to identify candidate patients. Evidence of clinical benefit by Id vaccination has been obtained so far only immunization with whole Ig protein vaccine [2,3], thus displaying to the host immune system both tumor-specific V H and V L .Therefore, considering that IGHV1-69 is often the partner of IGKV3-20 in HCV-related NHLs [18,20], a prototypic BCR for a subset of HCV-associated NHL was cloned, inserted into VB format and used to DNA immunize mice.We chose IGHV and IGKV from unrelated lymphomas purposely considering the possible use of such Vaccibodies as off-the-shelf, subgroup-specific vaccines.In fact, while HCV-associated NHL express IGHV1-69 and IGKV3-20 proteins with high similarity in the framework regions, the presence of several differences in the amino acid sequence of the CDR regions makes it difficult to select a one-for-all IGHV-IGKV pair.On these grounds, we selected IGHV1-69 and IGKV3-20 proteins as prototypic on the basis of their representativity of the FR regions among HCV-associated lymphomas.Our results show that such an artificial targeted DNA Id vaccine elicits antibodies in mice that bind mouse B lymphoma cells transfected with the H and L chain genes composing the artificial BCR.Whether these antibodies bind human IGHV1-69/IGKV3-20 B lymphomas remains to be investigated pending sample availability. From a translational standpoint, the possibility of using DNA vaccines encoding scFv with the potential to elicit cross-reactive immune responses is not restricted to the IGHV1-69/IGKV3-20 combination, as similar features of conserved V regions usage have been detected in other B cell malignancies.Thus, one can envision tailored Id vaccines for each major stereotyped subset identified.To estimate the number of patients with B-cell malignancies that could be immunized with off-the-shelf cross reactive vaccines, a large database including sequences of idiotypic VH and VL genes expressed by low grade B-NHL, autoimmunity-associated lymphoproliferations (e.g.HCV-related NHL, mixed cryoglobulinemia, Sjögren's syndrome) and CLL is currently being set up with the aim of identifying subgroups of tumors characterized by the expression of molecularly correlated Id proteins on the basis of the degree of sequence conservation among patients (R Dolcetti, unpublished results).However, stereotyped BCR sequences appear to be diseasebiased.In CLL, shared V regions are in most cases unmutated [25,26,48,49] whereas in other B cell tumors (e.g., HCV-associated lymphomas, MALT lymphomas) somatic mutations are more frequent [18,20,21].Therefore, from an immunological standpoint, the yet unanswered question as to whether the host immune system can recognize V region sequences in germline configuration following Id vaccination is of paramount relevance for the possibility of applying Id vaccines, whether individual or subgroup-specific [50]. It should be stressed that certain human B cell malignancies have not been described to express stereotyped BCR.One example is multiple myeloma cells that carry high loads of somatic mutations in their V regions, consistent with an origin from post germinal center B cells [51].In a previous report, we demonstrated that mice DNA-immunized with hybrid mouse/human Vaccibodies expressing scFv of either of four myeloma patients induced anti-Id antibodies that bound the corresponding protein with little cross-reactivity despite the fact that the BCR of two patients used the same IGHV and IGHJ genes [16].Thus, in the case of multiple myeloma, V regions of monoclonal Ig express unique Ids with little cross-reactivity, at least as defined by antibodies elicited by APC-targeted DNA Id vaccines. The discussion above has focused on antibodies elicited by APC-targeted DNA Id vaccines since anti-Id antibodies have been linked to anti-lymphoma activity both in passive and active immunotherapy [52,53].However, MHCrestricted, Id-specific T cells have been shown to display anti-lymphoma activity and to eradicate B cell tumors [43,47,54,55].scFv in the vaccine contains Id-sequences available for MHC presentation.T cell responses were not investigated in this study as they would have been directed against xenogeneic Ig sequences thus having no semblance to the clinical situation.Nevertheless, it can be speculated that subgroup-specific vaccines can elicit cross-reactive Id-specific T cell responses which may or may not be accompanied by cross-reactive humoral responses, as has recently been suggested with HCVassociated B cell lymphoma IGKV3-20 [56], However, patients differ in polymorphic HLA molecules and are therefore expected to present different sequences of V regions of CLL/B lymphoma BCR on their HLA molecules, thus making the possibility of cross-reactive T cell responses less common as compared with antibody responses.Also, based on mouse studies, T cell tolerance to the CLL/B lymphoma BCR is likely to limit T cell responsiveness in humans to a greater extent than humoral responses. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Herein we demonstrate the feasibility of constructing fully human Vaccibodies that target scFv Id to mouse APC in vivo enabling enhanced immune responses.The striking similarity of amino acid Id sequence found across different subgroups of patients affected by molecularly identified B cell malignancies could be exploited to prepare subgroup-specific vaccines.As opposed to patientspecific vaccines, such \"off-the-shelf\" vaccines could reduce the number of tailor-made Id DNA vaccines allowing substantial time and cost savings. ",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "The excellent technical help of Elisabeth Lea Vikse is gratefully acknowledged.Keith M Thompson established heterohybridoma to obtain CLL Ig.We are also grateful to personnel of the Institute for Comparative Medicine and the Flow Cytometry facility at Rikshospitalet, Oslo, for animal care and cell sorting, respectively.The study was supported by grants from the European Community (FP6 037874) and Associazione Italiana per la Ricerca sul Cancro (AIRC) contract 14287 to RD. PAR was a research fellow of the Norwegian Cancer Society. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Ab: Antibody; Ag: Antigen; APC: Antigen-presenting cells; BCR: B Cell receptor; CLL: Chronic lymphocytic leukemia; Fab: Fragment antigen binding; Id: Idiotype; Ig: Immunoglobulin; HCV: Hepatitis C virus; HLA: Human Leukocyte Antigen; MHC: Major histocompatibility complex; NHL: Non Hodgkin's lymphoma; scFv: Single chain fragment variable; V: Variable; VB: Vaccibody. BB and PAR are inventors of Vaccibody patent applications filed by their employer (Univeristy of Oslo and Oslo University Hospital).BB is head of the scientific panel of the company Vaccibody AS. Authors' contributions PAR, BB and RD conceived of the study.PAR carried out the construction and in vitro characterization of the vaccines, performed the mouse immunization studies, and drafted the manuscript.AO and LAM contributed to the CLL analysis and experiments.GET provided CLL patient samples, V region sequencing and clinical information.RD provided the plasmids for HCV-associated NHL BCR Vaccibody and helped to draft the manuscript.BB participated in the study design and coordination and helped to draft the manuscript.PAR, LAM and BB wrote the final manuscript.All authors read and approved the final manuscript. ",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "Ab: Antibody; Ag: Antigen; APC: Antigen-presenting cells; BCR: B Cell receptor; CLL: Chronic lymphocytic leukemia; Fab: Fragment antigen binding; Id: Idiotype; Ig: Immunoglobulin; HCV: Hepatitis C virus; HLA: Human Leukocyte Antigen; MHC: Major histocompatibility complex; NHL: Non Hodgkin's lymphoma; scFv: Single chain fragment variable; V: Variable; VB: Vaccibody. ",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "BB and PAR are inventors of Vaccibody patent applications filed by their employer (Univeristy of Oslo and Oslo University Hospital).BB is head of the scientific panel of the company Vaccibody AS. Authors' contributions PAR, BB and RD conceived of the study.PAR carried out the construction and in vitro characterization of the vaccines, performed the mouse immunization studies, and drafted the manuscript.AO and LAM contributed to the CLL analysis and experiments.GET provided CLL patient samples, V region sequencing and clinical information.RD provided the plasmids for HCV-associated NHL BCR Vaccibody and helped to draft the manuscript.BB participated in the study design and coordination and helped to draft the manuscript.PAR, LAM and BB wrote the final manuscript.All authors read and approved the final manuscript. ",
"section_name": "Competing interests",
"section_num": null
}
] |
10.1186/s13046-024-03210-9
|
Targeting cancer-associated fibroblasts/tumor cells cross-talk inhibits intrahepatic cholangiocarcinoma progression via cell-cycle arrest
|
<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>Cancer-associated fibroblasts (CAFs), mainly responsible for the desmoplastic reaction hallmark of intrahepatic Cholangiocarcinoma (iCCA), likely have a role in tumor aggressiveness and resistance to therapy, although the molecular mechanisms involved are unknown. Aim of the study is to investigate how targeting hCAF/iCCA cross-talk with a Notch1 inhibitor, namely Crenigacestat, may affect cancer progression.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>We used different in vitro models in 2D and established new 3D hetero-spheroids with iCCA cells and human (h)CAFs. The results were confirmed in a xenograft model, and explanted tumoral tissues underwent transcriptomic and bioinformatic analysis.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>hCAFs/iCCA cross-talk sustains increased migration of both KKU-M213 and KKU-M156 cells, while Crenigacestat significantly inhibits only the cross-talk stimulated migration. Hetero-spheroids grew larger than homo-spheroids, formed by only iCCA cells. Crenigacestat significantly reduced the invasion and growth of hetero- but not of homo-spheroids. In xenograft models, hCAFs/KKU-M213 tumors grew significantly larger than KKU-M213 tumors, but were significantly reduced in volume by Crenigacestat treatment, which also significantly decreased the fibrotic reaction. Ingenuity pathway analysis revealed that genes of hCAFs/KKU-M213 but not of KKU-M213 tumors increased tumor lesions, and that Crenigacestat treatment inhibited the modulated canonical pathways. Cell cycle checkpoints were the most notably modulated pathway and Crenigacestat reduced CCNE2 gene expression, consequently inducing cell cycle arrest. In hetero-spheroids, the number of cells increased in the G2/M cell cycle phase, while Crenigacestat significantly decreased cell numbers in the G2/M phase in hetero but not in homo-spheroids.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>The hCAFs/iCCA cross-talk is a new target for reducing cancer progression with drugs such as Crenigacestat.</jats:p> </jats:sec><jats:sec> <jats:title>Graphical abstract</jats:title> </jats:sec>
|
[
{
"section_content": "In the last forty years, the incidence of intrahepatic Cholangiocarcinoma (iCCA) in the U.S. has increased by 128% [1].Nevertheless, diagnostic skills, therapies and clinical management, responsible for prognosis and survival, have not significantly improved, and only approximately 20-30% of patients with iCCA are eligible for surgery [2], while most cases relapse, and best overall 5-year survival is not superior to 20% [3].This makes iCCA a highly deadly disease, and, the lack of a reliable drug-based therapy for patients at an advanced stage of disease is a severe drawback for patients and clinicians.For these patients, gold standard therapy is still based on Gemcitabine-Cisplatin, Gemcitabine-Oxaliplatin (GEMOX) and 5-Fluorouracil (5-FU) [4][5][6].However, chemotherapy is often only palliative, and this has driven the investigation of different targeted biological therapies.For instance, fusions at the Fibroblast Growth Factor Receptor gene (FGFR2) are reported in 10-15% of iCCA patients, for whom selective FGFR Tyrosine Kinase Inhibitors (TKIs) have been successfully tested, emphasizing once more the concept of precision medicine in iCCA [7,8].Overall, very little is known regarding the molecular mechanisms underlying iCCA progression, aggressiveness, and resistance to common chemotherapies. The iCCA hallmark is the desmoplastic reaction, characterized by an abundant extracellular matrix (ECM) deposition, creating tissue boundaries that provide structural and biochemical support and leading to drug resistance [9][10][11][12].Human Cancer Associated Fibroblasts (hCAFs) are a heterogeneous population of fibroblasts originating from various sources of resident fibroblasts, including stellate cells, that are mainly activated by Transforming Growth Factor (TGF)-1 and Platelet Derived Growth Factor (PDGF) that undergo the epithelial-to-mesenchymal transition (EMT), taking on a myofibroblast phenotype characterized by α-Smooth Muscle Actin (α-SMA) and Fibroblast Activation Protein (FAP) expression [13,14].hCAFs are mainly responsible for the ECM deposition and for remodeling the surrounding tissue microenvironment [15]. hCAFs establish a dynamic and mutual cross-talk with epithelial cancer cells, and are believed to contribute to tumor progression in different malignancies including iCCA [16][17][18].To date, cytokines, growth factors, miRNA, freely released or carried by microvesicles are likely involved, but the mechanisms triggered by such a cross-talk are still unclear. Notch pathway activation is triggered by direct cellto-cell communication between Jagged (JAG 1, 2) or Delta-like (DLL 1, 3, and 4) ligands and Notch receptors (1-4), followed by cleavage of the Notch transmembrane domain by ɣ-secretase that leads to regulation of the transcription of target genes involved in cell proliferation, differentiation, and cell death.Aberrant Notch cleavage by ɣ-secretase has recently been demonstrated to be a main driver in the initiation and progression of iCCA [19].Recently, we reported that Crenigacestat, a gamma secretase inhibitor, targets the NOTCH1/DLL4/ VEGFA/MMP13 axis, reducing iCCA progression in a patient-derived xenograft (PDX) model by blocking tumor neovascularization [20].We also demonstrated that Crenigacestat inhibits iCCA tumor progression in NOTCH1/HES1/THY1-positive xenograft models [21].Moreover, in the surrounding ecosystem of iCCA, Crenigacestat inhibits the occurrence of liver fibrotic reactions by deactivating and reverting the myofibroblastic phenotype of iCCA hCAFs to a fibroblastic phenotype with a reduced secretion of TGF-β and ECM components such as FN, COL1A1, and COL1A2 [22]. The aim of this study is to investigate whether the hCAFs/iCCA crosstalk may be a suitable target for drugs such as Crenigacestat, and whether this could affect iCCA cancer progression. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To investigate the role of the CAF/iCCA cells cross-talk, we challenged KKU-M213 and KKU-M156 iCCA cell lines to migrate in the presence of human hCAFs, isolated from three different patients who underwent surgery for iCCA.As reported in Fig. 1A-B, both KKU-M213 and KKU-M156 cell lines migrated on Coll I, more efficiently (p < 0.001 and p < 0.01) in the presence than absence of hCAFs seeded at the bottom part of the transwell.Consistently, the cell viability viability and proliferation of both KKU-M213 and KKU-M156, Fig. 1C-DB, was significantly (p < 0.01 and p < 0.001 for viability; and p < 0.05 for proliferation) increased in the presence compared to the absence of hCAFs.In conclusion, the hCAFs/iCCA cells cross-talk promoted the migration, viability and proliferation of iCCA cells. ",
"section_name": "Effects of CAFs/iCCA cells cross-talk on migration and viability of iCCA cells",
"section_num": null
},
{
"section_content": "To investigate the effectiveness of Crenigacestat on the hCAFs/iCCA cells cross-talk, we treated each cell component individually, and then combined them for challenging iCCA cells to migrate.Firstly, we treated both iCCA cell lines with Crenigacestat or vehicle under the same experimental conditions previously described; we seeded them in the upper part of the insert coated at the bottom part with Coll I, and challenged them to migrate for 18 h in fresh medium without any drug but in the presence of hCAFs seeded at the bottom part of the transwell.As reported in Supplementary Fig. 1, the treatment with Crenigacestat did not affect cell migration of both iCCA cell lines as compared to the treatment with vehicle.Secondly, in a different set of experiments, we treated hCAFs cultured at the bottom part of the transwell with Crenigacestat or vehicle under the same experimental conditions previously described, and challenged both iCCA cell lines seeded on the top of the insert coated at the bottom part with Coll I to migrate for 18 h in the presence of fresh medium without any drug.As reported in Supplementary Fig. 2, also in this case Crenigacestat did not affect cell migration as compared to vehicle.Thirdly, in a further set of experiments, we treated separately both iCCA cell lines and hCAFs with Crenigacestat and vehicle in different wells, then we allowed iCCA cells lines to migrate for 18 h on filters coated with Coll I in the absence of any drug but in the presence of hCAFs, previously seeded.As shown in Supplementary Fig. 3, also in this case Crenigacestat treatment did not affect cell migration.Finally, we treated cells for 72 h with Crenigacestat or vehicle KKU-M213 as well as KKU-M156 cells cultured on the top of the insert and hCAFs at the bottom of the transwell at the same time.Then, iCCA cells were trypsinized, washed and seeded on the top of a new insert coated at the bottom part of the filter with Coll I, and challenged to migrate for 18 h in fresh medium without any drug, in the presence of the previously treated hCAFs.As reported in Fig. 2A-B, Crenigacestat significantly inhibited KKU-M213 and KKU-M156 cell migration (p < 0.001 and p < 0.01, respectively) as compared to vehicle, but it did not affect cell viability and proliferation using all the same hCAF preparation in 2D models (Fig. 2C-D).All the experimental conditions were run in experimental triplicate, and each experiment was re peated in biological triplicate using preparations of hCAFs isolated from three different patients who underwent surgery for iCCA. In conclusion, Crenigacestat inhibits iCCA cell migration only when the hCAFs/iCCA cell cross-talk occurs. ",
"section_name": "Crenigacestat effectiveness on migration induced by the hCAFS/iCCA cells cross-talk",
"section_num": null
},
{
"section_content": "To investigate in further depth the role of the hCAFs/ iCCA cells cross-talk we developed a three-dimensional (3D) in vitro model shaped by only iCCA cells (homo-spheroids) or by hCAFs and iCCA cells (heterospheroids), in which both cell types are in tight contact.Briefly, 3D-structures formed exclusively by iCCA cells or by both hCAF and iCCA cells were characterized by immunophenotyping using Epithelial Cell Adhesion Molecule (EpCAM) and Fibroblast Activation Protein (FAP) expression, as well-known markers of iCCA and hCAFs cells, respectively.As expected, immunofluorescence staining revealed EpCAM-positive expression in homo-and hetero-spheroids of KKU-M213 and KKU-M156, while FAP-positive expression was positive only in hetero-spheroids, Fig. 3A and movies (Supplementary Video 1-4), thus confirming the presence of both iCCA cells and hCAFs.Then, homo and hetero-spheroids were stained with anti-Ki-67 antibodies, to explore cell proliferation activity.As described in Fig. 3B and movies (Supplementary Video 5-8), the number of Ki-67 positively stained cells was significantly (p < 0.001) higher in heterospheroids, 52.4% and 48.1% respectively for the presence of KKU-M213 and KKU-M156 with hCAFs versus 6.8% and 11.0% for the corresponding homo-spheroids. In conclusion, a cross-talk between hCAFs/iCCA cells increased proliferation. ",
"section_name": "Characterization of hCAFs/ iCCA cells in hetero-spheroid 3D structures",
"section_num": null
},
{
"section_content": "To investigate the effectiveness of Crenigacestat on the iCCA/hCAFs cross-talk modulating cellular viability and invasion in 3-D models, we bioengineered two distinct types of biological scaffolds formed by different extracellular matrix proteins enriched with hydrogel to better support cell viability or cell invasion, respectively.Firstly, we cultured, for 96 h, KKU-M213 or KKU-M156 hetero-spheroids embedded in the proliferative permissive hydrogel, whereby significantly (p < 0.01 and p < 0.01, respectively), they doubled the surface area and showed a significantly (p < 0.01 and p < 0.05, respectively) increased viability compared to the homo-spheroids, see Supplementary Fig. 4 and Fig. 4A.Under the same experimental conditions previously described, we tested homo-and hetero-spheroids with Crenigacestat, and after 96 h, the drug treatment significantly reduced KKU-M213 and KKU-M156 hetero-spheroids viability (p < 0.001 and p < 0.01 respectively) as compared to vehicle.Consistently with the results described above, Crenigacestat treatment did not affect homo-spheroids viability (Fig. 4A). Next, we cultured homo-and hetero-spheroids in invasion-permissive hydrogel for 72 h.As observed in Fig. 4B, KKU-M213 and KKU-M156 hetero-spheroids significantly invaded the surrounding area (p < 0.05 and p < 0.01, respectively) as compared to homo-spheroids.Furthermore, Crenigacestat treatment significantly inhibited the invasion of KKU-M213 and KKU-M156 heterospheroids (p < 0.05 and p < 0.01, respectively), but not of homo-spheroids, Fig. 4B.All the experiments of 3D model viability and invasion were performed in technical triplicate and in biological triplicate using preparations of hCAFs isolated from three different iCCA patients. In conclusion, this bulk of data suggests that the iCCA/ hCAFs crosstalk plays a key role in the growth and invasion of hetero-spheroids.Consistently, Crenigacestat reduces cell viability and invasion only in the presence of the iCCA/hCAFs crosstalk. ",
"section_name": "Crenigacestat inhibits the viability and invasion of heterospheroids",
"section_num": null
},
{
"section_content": "To better investigate the hCAFs/iCCA cells cross-talk in vivo, we generated a xenograft model by injecting KKU-M213 exclusively or in combination with hCAFs but normalizing for the total number of cells.Tumors originated by KKU-M213 and hCAFs grew larger as compared with those originated by KKU-M213 alone, and this difference was already significant (p < 0.02) after 10 days, and persisted until the end of the experiment.Consistently with all the in vitro results, Crenigacestat significantly (p < 0.05) reduced the volume of the hCAFs/KKU-M213 tumors after 10 days of treatment and maintained this effect until the end of treatment, showing an even more significant difference (p < 0.02) when compared to untreated mice.On the contrary, no statistically significant effects were observed on tumors generated by KKU-M213 alone during the whole treatment (Fig. 5A-B). To confirm that our xenograft model reliably resembles a human iCCA, we stained sections from KKU-M213 and KKU-M213/hCAFs tumors with an anti-CK-19 antibody.As reported in Fig. 5C, CK-19, a specific marker of CCA, was diffusely expressed, showing a membranous and cytoplasmic homogenous pattern.A smaller but still relevant number of tumor cells displayed consistent cytokeratin 7 immunoreactivity (data not shown).Following Masson trichrome staining, all sections showed variable amounts of intercellular collagenous matrix.In KKU-M213 tumors, it appeared as thin light blue bundles surrounding the periphery of tumor cell clusters, and including scarce and sparse fibroblasts, lympho-monocytes, and neutrophilic granulocytes.No changes were observed after Crenigacestat treatment.On the contrary, in KKU-M213/hCAFs tumors the extracellular collagen bundles were much larger, more intensely stained in blue, and included more numerous fibroblasts, which were also more tightly packed.After treatment with Crenigacestat, collagen bundles were less evident around the tumor clusters and they showed a lighter bluish discoloration and included smaller amounts of fibroblasts inside.Furthermore, we quantified the amount of the deposited fibrotic matrix using an adapted METAVIR score, as previously reported [22].Liver fibrosis was significantly (p < 0.001) more abundant in KKU-M213/hCAFs than in KKU-M213 tumors, where it was organized as reactive tumoral stroma.Crenigacestat treatment significantly (p < 0.001) reduced liver fibrosis on KKU-M213/hCAFs compared to KKU-M213 tumors, but it did not display any effect on other models, Fig. 5C. In conclusion, the co-injection of both hCAFs and KKU-M213 cells forms bigger tumors, but they are druggable with Crenigacestat. ",
"section_name": "Crenigacestat reduces the fibrosis and growth of hCAFs/ iCCA tumors in vivo",
"section_num": null
},
{
"section_content": "To investigate the molecular mechanisms triggered by the hCAF/KKU-M213 cross-talk in the xenograft mice models with and without Crenigacestat treatment, we performed a transcriptomic analysis on tissues explanted from xenograft mice (n = 3/group).Firstly, we compared the tumor masses derived from co-injected KKU-M213 and hCAFs with those derived from only KKU-M213.This comparison identified 129 DEGs between the two groups, and the expression profiles of these DEGs were visualized through principal component analysis (PCA) and the hierarchical clustering heatmap (Fig. 6A).Then, we verified the molecular changes induced by Crenigacestat in the xenograft model derived from co-injected KKU-M213 and hCAFs, and from KKU-M213 only.We detected 554 genes differentially expressed between the two groups.PCA and hierarchical clustering heatmap showed a clear separation between the two groups (Fig. 6B). To gain insight into the biological effects of DEGs, we performed an IPA core analysis.The IPA annotations \"Disease & Function\" revealed that the most numerous genes for both comparisons were present in Hepatic System Diseases.In particular, based on this analysis, genes from co-injected hCAFs and KKU-M213 tumors were able to increase the formation of Liver lesions and Liver tumors (Fig. 6C).Contrariwise, the treatment of Crenigacestat in this mice model reduced the formation of Liver lesions and Liver tumors.Other biological functions affected by Crenigacestat were Hepatobiliary carcinoma, Hepatobiliary system cancer, Cholangiocarcinoma, Liver cholangiocarcinoma and Growth (Fig. 6D).Next, we evaluated the modulated canonical pathways that led to a reduction of liver lesions after Crenigacestat treatment in the xenograft model derived from co-injected KKU-M213 and hCAFs.We focused our attention on the genes that are involved in the function Liver lesions, which numbered 280 of the total 554, and we found that in the top 20 modulated pathways, several signaling pathways controlled the cell cycle progression (Fig. 7A).Specifically, 37 differentially expressed genes (DEGs) were modulated by Crenigacestat and regulated the cell cycle checkpoints (Fig. 7B).Of note, several genes that encode for cyclins (CCNA2, CCNB1, CCNB2, CCNE2) were downregulated after Crenigacestat treatment.Among these genes, we hypothesized that Crenigacestat affected CCNE2 expression that, in turn, reduced the expression of downstream cyclins like CCNA2, CCNB1, CCNB2 (Fig. 7C).To validate the sequencing results, we performed quantitative real-time PCR for CCNE2 on masses of the xenograft model derived from co-injection of KKU-M213 and hCAFs.We found that, after Crenigacestat treatment, the mRNA expression of CCNE2 significantly decreased compared to untreated masses (p < 0.001, Fig. 7D) suggesting that cell cycle progression could be arrested in the G1 phase.As control, we also verified the expression of CCND1 that controls the previous phase of cell cycle and we observed that Crenigacestat treatment did not have any influence on its expression (Fig. 7D). In conclusion, IPA core analysis revealed that genes from tumors with a hCAFs/KKU-M213 cross-talk had increased liver lesions, and that this was decreased by Crenigacestat treatment.Finally, cell cycle progression was the most notably modulated pathway following Crenigacestat treatment. ",
"section_name": "Transcriptomic analysis of explanted tumor masses",
"section_num": null
},
{
"section_content": "To validate the transcriptomic and bioinformatic results previously described, we investigated Crenigacestat effectiveness on cell cycle homo and hetero-spheroids.Analyzed by flow cytometry, consistently with all previous data, the hCAFs/iCCA cells cross-talk significantly (p < 0.05) increased the cell numbers in the G2/M phase compared to the homo-spheroids, leading to a greater growth of hetero-spheroids.Next, both homo and hetero-spheroids were treated with Crenigacestat 5 µM for 96 h.As reported in Fig. 8, Crenigacestat significantly (p < 0.01) arrested the cell population of the hetero-spheroids in the G0/G1 cell cycle phase, reaching 43.15%, while with vehicle treatment it reached 30.76%.Consistently, the G2/M phase in hetero-spheroids significantly (p < 0.05) decreased to 53.16% after treatment with Crenigacestat, while after vehicle treatment it reached 60.13%.Finally, Crenigacestat did not display any effect on homo-spheroids.All the experiments were run in triplicate using three different preparations of hCAFs isolated from distinct iCCA patients who underwent surgery. In conclusion, the hCAFs/iCCA cells cross-talk increased the growth of hetero-spheroids, increasing cells in the G2/M cell cycle phase, while Crenigacestat treatment decreased the growth of hetero-but not homo-spheroids, inducing a decrease of the number of cells in the G2/M cell cycle phase. ",
"section_name": "Crenigacestat arrests the cell cycle at the G0/G1 phase in hetero-spheroids",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "KKU-M213 and KKU-M156 human iCCA cell lines, purchased from the Japanese Collection of Research Bioresources (JCRB) or the American Type Culture Collection (ATCC), were used for the experiments.Cell Lines Service (Eppelheim, Germany) performed cell line authentication.Both cell lines were grown in Dulbecco's modified Eagle medium (DMEM), (Gibco, Grand Island, NY, USA) supplemented with 10% Fetal Bovine Serum (FBS), Antibiotic-Antimycotic, Sodium Pyruvate and Hepes, (Gibco, Grand Island, NY, USA).Cells were cultured at 37 °C in a 5% CO 2 humidified atmosphere.Cell lines, before use, were tested mycoplasma-free using the Myco-Fluor™ Mycoplasma detection Kit (ThermoFisher Scientific, Waltham, MA, USA).Crenigacestat (LY3039478, Selleckchem Chemicals, Houston, TX, USA) was used to treat iCCA cell lines in vitro and in animal models in vivo.Stock solutions were prepared in Dimethylsulfoxide (DMSO) (ThermoFisher Scientific, Waltham, MA, USA) and aliquots were stored at -80 °C. ",
"section_name": "Cell lines and reagents",
"section_num": null
},
{
"section_content": "Approval of the study was granted by the local Ethics Committee, Istituto Tumori \"Giovanni Paolo II\" (Bari, After surgical resection, iCCA tissue specimens were immediately stored in MACS tissue storage solution (Miltenyi Biotec, Bergisch Gladbach, Germany) and processed for hCAFs isolation as previously reported [23].iCCA hCAFs were isolated from three patients.We performed enzymatic and mechanical digestion of iCCA tissue fragments in HBSS solution with 50-200 U/mL collagenase Type IV (Thermo Fisher Scientific, Waltham, MA, USA), 3 mM CaCl 2 , and Antibiotic-Antimycotic (Thermo Fisher Scientific, Milan, Italy) at 37 °C by rotation for 2 h or more as needed.The resulting cells were harvested by recovering the volume of digestion and washed with PBS.The cell population harvested was cultured in complete IMDM (Iscove's Modified Dulbecco's Medium) with 20% FBS and Antibiotic-Antimycotic at 37 °C in a 5% CO 2 humidified atmosphere to obtain hCAF cultures. ",
"section_name": "Establishment of iCCA hCAF culture from fresh human iCCA tissues",
"section_num": null
},
{
"section_content": "For the co-culture, 15 × 10 3 iCCA cells were seeded onto 24-well plates with complete DMEM medium.Additionally, 20 × 10 3 hCAFs were seeded onto transwell inserts with 0.4 μm pore size (Corning, Bedford, MA, USA) in complete IMDM medium.After 24 h, the transwell inserts with seeded hCAFs were transferred to 24-well plates for co-culturing with iCCA cells.As control, iCCA cells without hCAFs in transwell inserts were prepared.iCCA cell cultures and hCAFs/iCCA co-cultures prepared in this way were treated with vehicle and Crenigacestat (5 µM) in IMDM + 1% FBS for 72 h.At the end of treatment, iCCA cell viability in the presence or absence of hCAFs was determined with the CyQUANT™ XTT Cell Viability kit (ThermoFisher Scientific, Waltham, MA, USA) and for proliferation by Trypan Blue cell counting.Data are expressed as the mean ± SD of three independent experiments with three hCAFs. ",
"section_name": "Viability and proliferation assay on iCCA cell in co-culture with hCAFs",
"section_num": null
},
{
"section_content": "KKU-M213 or KKU-M156 cell migration was induced for 18 h, after 72 h of treatment.In detail, we developed three different co-culture approaches: pre-treating for 72 h 15 × 10 3 iCCA cells onto transwell inserts with 0.4 μm pore size or 40 × 10 3 hCAFs onto 24-well plates separately, or both cell types simultaneously in co-culture with Crenigacestat (5 µM) or vehicle.In each case, after the treatment, we evaluated the iCCA cell migration in co-culture with 40 × 10 3 hCAFs.As described for the vitality assay, also for the study of this cell functional aspect, we used a control condition, namely the migration of iCCA cells without hCAFs seeded in the bottom of the wells.Cell migration was allowed by using transwell inserts suitable for 24-well plates, with 6.5 mm internal diameter, and 8 μm pore size (Corning, NY, USA).The membrane of these transwell inserts had been coated with rat tail collagen I (final concentration 10 µg/ mL) on the lower surface for 2 h at room temperature.20 × 10 3 iCCA cells were suspended in 200 µL of serumfree IMDM medium and loaded onto the top chamber of transwell inserts.Cells were allowed to migrate for 18 h in IMDM medium + 10% FBS at 37 °C and 5% CO 2 .After incubation, the migrated cells were fixed in 4% PFA (pH 7.2 in PBS) and stained with crystal violet for 10 min.Five fields per membrane were captured in bright field at 10× magnification.The analysis was performed on the mean number of migrated cells/field.Data are expressed as the mean ± SD of three independent experiments with three hCAFs. ",
"section_name": "Transwell migration assay",
"section_num": null
},
{
"section_content": "Three-dimensional (3D) cell culture models were set up using the hanging drop method as previously described [24].Briefly, cells were suspended, at a concentration of 1 × 10 5 cells/ml, in medium with 0.24% methylcellulose (Sigma, St. Louis, MO, USA).Forty drops of 25 µl each were pipetted onto the lid of 100 mm dishes.In this way each drop and thus each spheroid consisted of 2.5 × 10 3 cells.Homo-spheroids were developed starting from a single cell type, KKU-M213 or KKU-M156 cells.Instead, hetero-spheroids were developed by respecting the 1:3 rate for KKU-M213 or KKU-M156 cells: hCAFs.After 3 days of incubation at 37 °C and 5% CO 2 , spheroids were transferred into different culture supports depending on the cell function assay.Each experimental condition was performed three times and hetero-spheroids were replicated with three hCAFs.Values are presented as mean ± SD. ",
"section_name": "3D homo-spheroid and hetero-spheroid cultures",
"section_num": null
},
{
"section_content": "For cell cycle analysis, the recovered spheroids were cultured on an ultralow-attachment culture plate (Corning, Bedford, MA, USA) and treated with vehicle or Crenigacestat (5µM) twice for 5 days.The cell cycle was induced on cell suspensions obtained after disruption of the homo-and hetero-spheroids.Briefly, floating spheroids were disrupted using Tryple™ Select (Gibco, life Technologies corporation, NY, USA) plus 1mM EDTA pH 8.0 (Invitrogen by ThermoFisher Scientific, USA) and pipetting every 10 min. 1 × 10 6 cells were washed once in cold PBS and fixed with cold 70% ethanol at 4 °C overnight.The fixed cells were then washed twice in PBS, the supernatant was discarded, and the cells were treated with of RNase A (Sigma, United States; 100 µg/mL) for 15 min at 37 °C.Propidium iodide; Sigma, United States; 200 µL of 50 µg/mL stock) was then added to the cells and incubated for 30 min at 4 °C in the dark.The DNA content of the cells was determined by flow cytometry. ",
"section_name": "Flow Cytometric analysis of cell cycle",
"section_num": null
},
{
"section_content": "For the viability assay, the spheroids were embedded in engineered hydrogel networks made of bovine skin hydrolyzed collagen and Corning® Matrigel® Matrix; the pH was neutralized with 0.5 M acetic acid.To prevent the spheroids from settling and sticking to the bottom of the well, a drop of matrix was placed in the well and allowed to dry for 20 min before embedding the samples into the matrix.In addition, spheroids were treated with vehicle and Crenigacestat (5µM).After 5 days of treatment, cell viability was assayed using the CellTiter-Blue™ Viability Assay (Promega, Tokyo, Japan). ",
"section_name": "Spheroid viability assay",
"section_num": null
},
{
"section_content": "The spheroids were embedded in an engineered hydrogel network.The bovine collagen type I solution and Corn-ing® Matrigel® were mixed and neutralized with 0.5 M acetic acid to obtain a matrix suitable for the invasion assay.Fresh medium was added to the final gel solution, to simulate cell embedding.Spheroids were treated with Crenigacestat (5µM) or vehicle for 72 h.Images were captured in bright field at 10× magnification.The analysis was performed on the area of invasion ratio of treated spheroids/untreated spheroids. ",
"section_name": "Spheroid invasion assay",
"section_num": null
},
{
"section_content": "For immunofluorescence staining, the spheroids were fixed in a 4% PFA solution at 4 °C for 30 min, and then washed twice in 0.1% Triton X-100 in TBS.After washing, samples were permeabilized with 0.5% Triton X-100 in TBS at room temperature.Then, the spheroids were incubated with anti-FAP (1:150, Abcam, Cambridge, UK), anti-EPCAM antibodies (1:800, Cell Signaling Technologies, MA, USA) in an antibody dilution buffer (2% bovine serum albumin/0,1% Triton X-100 in TBS solution) and anti-Ki67 (1:250, Abcam, Cambridge, UK), overnight at 4 °C in rotation.The following day, after washing, samples were incubated with secondary goat anti-rabbit immunoglobulin G H&L (1:500 Alexa Fluor 594, Thermo Fisher Scientific, Waltham, MA, USA) for FAP, with secondary goat anti-mouse immunoglobulin G H&L (1:50 Alexa Fluor 488, Thermo Fisher Scientific, Waltham, MA, USA) for EPCAM and with secondary goat anti-rabbit immunoglobulin G H&L (1:50 Alexa Fluor 488, Thermo Fisher Scientific, Waltham, MA, USA) for Ki67.The incubation with secondary antibodies lasted 3 h at room temperature in the dark.After further washing, nuclei staining was performed with 0.5 ng/ml of PureBlu DAPI Nuclear Staining Dye (Bio-Rad Laboratories, USA) in 0.1% Triton X-100 in TBS, incubating for 1 h at room temperature in the dark.Spheroids were put on the slides and covered with ProLong™ Diamond antifade mounting medium (Invitrogen by ThermoFisher Scientific, USA).Confocal images and movies of spheroids were acquired using the Nikon Ti2-E Inverted Research Microscope equipped for confocal imaging in conjunction with a Nikon A1rSi Laser Point Scanning Confocal System, Plan Fluor Ph 20X objective and NIS-Elements \"AR\" 5.0 software and improved by deconvolution method based on Richardson-Lucy Algorithm. ",
"section_name": "Spheroid immunofluorescence",
"section_num": null
},
{
"section_content": "Housing and all procedures involving the mice were performed according to the protocol approved by the Ethics Committee (Protocol number 257/2023-PR, date of release 29/03/2023) at Biogem Animal House in Ariano Irpino (Avellino, Italy) following the National Academy of Sciences Guidelines.Two million KKU-M213 cell lines or KKU-M213:hCAFs, respecting the 1:1 ratio, were subcutaneously injected into the flanks of 4-5-week-old female CD1 nude mice.Drinking water was supplied ad libitum.Each mouse was offered a complete daily pellet diet (GLP 4RF21, Mucedola) throughout the study.The analytical certificates of animal food and water were retained at Biogem premises.Each mouse was monitored daily for clinical signs and mortality, and body weight was recorded twice a week.The tumor volume was monitored weekly by a caliper and evaluated with the formula (mm 3 ) = [length (mm) × width (mm) 2 ]/2, where width and length are the shortest and longest diameters.When the tumor masses volume reached approximately 70-100 mm 3 , the mice were randomly subdivided into 2 experimental groups of six animals and administered Crenigacestat (8 mg/kg) or vehicle by oral gavage every 2 days for 20 days.At the end of the study, mice were sacrificed by cervical dislocation, and tumor samples were collected and sectioned for immunohistochemical, hematoxylin-eosin and trichrome staining or for RNA extraction. ",
"section_name": "In vivo study",
"section_num": null
},
{
"section_content": "Tumor specimens were fixed in 4% paraformaldehyde and embedded in paraffin using standard procedures.To analyze the grade of tissue fibrosis Masson's trichrome staining with the Mallory trichrome acc.McFarlane kit (DIAPATH) was performed, following the manufacturer's instructions.The degree of fibrosis was classified according to the adapted METAVIR score as previously reported [19].The images were acquired with the Eclipse Ti2 microscope (Nikon Inc., Melville, NY, USA). ",
"section_name": "Histological staining method",
"section_num": null
},
{
"section_content": "Total RNA from explanted tumor masses was isolated with the miRNeasy mini kit (Qiagen, Hilden, Germany) in combination with the TissueLyser homogenizer (Qiagen, Hilden, Germany) according to the manufacturer's instructions.The RNA concentration was determined with the Qubit™˝ฏ RNA HS Assay kit (Thermo Fisher Scientific, Waltham, MA, USA) on a Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA).RNA quality was evaluated using the High Sensitivity RNA ScreenTape (Agilent Technologies, Palo Alto, CA, USA) on an Agilent 4200 TapeStation system (Agilent Technologies). ",
"section_name": "RNA extraction",
"section_num": null
},
{
"section_content": "Total RNA samples were reverse transcribed using the Ion Torrent™ NGS Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.Target region amplification was performed using the Ion AmpliSeq Transcriptome Human Gene Expression core panel (Thermo Fisher Scientific, Waltham, MA, USA) on the Ion Chef System.The barcoded libraries were quantified by qPCR with the Ion Library TaqMan Quantitation kit (Thermo Fisher Scientific, Waltham, MA, USA).Finally, libraries were templated onto the Ion Chef and sequenced using a 540 chip on the Ion GeneStudio S5 Prime system (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing data are available under accession number GSE273905 at the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?&acc=GSE273905). ",
"section_name": "Whole transcriptome profiling",
"section_num": null
},
{
"section_content": "cDNA was reverse transcribed using the iScript Reverse Transcription Supermix (Bio-Rad Laboratories) according to the manufacturer's instructions.Quantitative PCR reactions were performed using SsoAdvanced SYBR green (Bio-Rad Laboratories) and the primers sequences for CCNE2 forward, 5 ",
"section_name": "Quantitative real-time PCR",
"section_num": null
},
{
"section_content": "A-3′, and Hs_GAPDH_1_SG QuantiTect Primer Assay ID: QT00079247 (Qiagen).The CFX96 System (Biorad, Hercules, CA, USA) was used for Real-Time PCR.Comparative real-time PCR was performed in triplicate, including no-template controls.Relative expression was calculated using the 2 -ΔΔCt method. ",
"section_name": "-T C A A G A C G A A G T A G C C G T T T A C-3′; reverse, 5-T G A C A T C C T G G G T A G T T T T C C T C-3′, CCND1 forward, 5-G C T G C G A A G T G G A A A C C A T C-3′; reverse, 5-C C T C C T T C T G C A C A C A T T T G A",
"section_num": null
},
{
"section_content": "Ion Torrent Suite Server v5.16.1 (Thermo Fisher Scientific, Waltham, MA, USA) software was used to generate the transcription data as raw read counts using the ampliSeqRNA plugin with default settings.Downstream analyses were performed with Transcriptome Analysis Console 4.0 software (Thermo Fisher Scientific, Waltham, MA, USA).DEGs were identified with the Limma eBayes method using a 1.5 threshold of fold-change and p-value ≤ 0.05.Hierarchical clustering was generated with Alt Analyze 2.1.3software [25].Canonical pathways, biological processes and molecular networks associated with DEGs were analyzed with Ingenuity Pathway Analysis (IPA) software (Qiagen, USA).Biological and technical replicates were analyzed with the most appropriate statistical tests (i.e.t test or ANOVA).For in vivo studies, Mann-Whitney U Test was performed.A p-value ≤ 0.05 was considered statistically significant.Statistical analysis and graphs were generated using GraphPad Prism 5.0 software (La Jolla, CA, USA). ",
"section_name": "Bioinformatics and statistical analyses",
"section_num": null
},
{
"section_content": "The reactive stroma surrounding and embedding the epithelial cancer cells characterizes the histological aspects of iCCA.CAFs secrete the ECM components of the stroma, and orchestrate constant tissue remodeling as a consequence of the further secretion of proteolytic enzymes, cytokines, and growth factors.Consequently, the biochemical composition and biological functions of the stroma are modified, so that cancer cells engage with ECM components, negotiating the expansion of tumor volume and spread at their best convenience [18,26,27].Based on these aspects, hCAFs are considered worldwide as an important component of the tissue microenvironment that directly or indirectly modulates the aggressiveness of cancer cells [28,29]. Nevertheless, clear cut evidence proving that the hCAFs/cancer cells crosstalk supports the progression of iCCA has never yet been reported.In this study we demonstrate that the growth of iCCA is stimulated by the tumor/stroma crosstalk, that in turn can be targeted with drugs such as Crenigacestat, leading to an inhibition of iCCA progression.We based our conclusion on the following data: (i) iCCA cells migrated and proliferated more efficiently only when in tight contact with hCAFs for a sufficient period to establish a cross-talk, (ii) Crenigacestat selectively inhibited iCCA cell migration stimulated by the cross-talk; (iii) the hetero-spheroids formed by hCAFs and the iCCA cell lines KKU-M213 or KKU-M156, by establishing a cross-talk, proliferated and invaded more efficiently than homo-spheroids formed by only KKU-M213 or KKU-M156 cells; Crenigacestat inhibited the growth and the proliferation of hetero-but not of homo-spheroids; iiii) in xenograft models, tumors generated by co-injection of both hCAFs and KKU-M213 cells are bigger than those generated by KKU-M213, while Crenigacestat inhibited the growth of the former but not of the latter tumors; iiiii) IPA core analysis of the transcriptomic investigation on tumoral masses of co-injected hCAFs and KKU-M213 cells pointed out that genes increased the formation of liver lesions, that was then inhibited by Crenigacestat; iiiiii) Crenigacestat downregulated Cyclin-E2 with a consequent arrest of the cell-cycle checkpoint in hCAF/KKU-M213 tumors, and in heterospheroids it arrested the cell cycle in the G2/M phase. The novelty of our study is the focus on targeting the tumor-stroma cross-talk, whereas other recent studies highlighted the effects consequent to targeting specific pathways such as hedgehog in pancreatic cancer.In this case, IPI-926 directly targeted hCAFs, affecting the number of ɑ-SMA positive cells, and therefore their capability of secreting ECM components.The study also shows that IPI-926 inhibitor rearranges the tissue microenvironment composition, reducing the deposition of Coll I, the surrounding fibrotic reaction, and increasing microvascular density and the delivery of chemotherapeutic agents [30]. On the other hand, the relevance of an increased prevalence of α-SMA+/CAFs in the stroma of iCCA was found to be correlated with a more malignant behavior and poorer survival outcomes in iCCA patients [31].Consistently, other studies identified CAFs as responsible for increasing the number of tumor-initiating cells in colorectal cancer, and targeted TGF-β1, also involved in CAFs activation, reducing cancer metastasis.However, also in this case drug treatment targeted stromal cells without any evident effect on the tumor/stroma crosstalk [32].Chimeric antigen receptors (CARs) T cells specific for FAP expressed on hCAFs have been reported to contribute to the therapeutic response in a lung tumor model, further supporting the idea of targeting CAFs because they are responsible for the deposition of the ECM components of the desmoplastic reaction [33].We also report that Crenigacestat changed the ECM composition of the surrounding tissue microenvironment in PDX models of iCCA [22].In accordance with our previous study, herein we also showed a reduction of the reactive stroma following Crenigacestat treatment, but in addition, we demonstrated that Crenigacestat inhibited the migration of iCCA cells only when both hCAFs and KKU-M213 and KKU-M156 were treated in cocultured conditions simultaneously for a sufficient time to establish a cross-talk.On the contrary, Crenigacestat did not affect cell migration when the drug was used to treat hCAFs and both the iCCA cell lines each time, without being co-cultured but assembled in the assay at the end of the treatment.This phase-by-phase experimental approach further supported the hypothesis that Crenigacestat effectiveness is downstream, acting by inhibiting the tumor/stroma cross-talk.The relevance of targeting the cross-talk was also supported by the effects of Crenigacestat, arresting the cell cycle in hetero-spheroids and in the bioinformatic and transcriptomic analysis of tumor masses.This further endorses our hypothesis whereby Crenigacestat displays its effect on hCAFs/iCCA cells, leading a downstream effect on cell proliferation, downregulating cyclin E2 and arresting the cell-cycle checkpoint.This is consistent with a previous study reporting cyclin E protein overexpression in iCCA, and hence cyclin E gene as a transcriptional target of Notch signaling [34].We hypothesize, based on our experimental data, that soluble factors released in the hCAF/iCCA cells cross-talk are likely involved in tumor progression.Therefore, the reduction of the fibrotic tissue we observed is only a part of the Crenigacestat effectiveness in targeting the hCAFs/iCCA cells cross-talk. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "In conclusion, we point out that the cross-talk between hCAFs and iCCA cells is a suitable target for drugs such as Crenigacestat but likely not only, thus opening out prospects for future investigations to develop new therapeutic strategies aimed at a precision medicine approach. ",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "The authors are grateful to Mary V.C. Pragnell, B.A., for the English revision and Giusy Bianco for technical support. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This study was supported by AIRC grant number IG 2020 ID 24815 and by Ricerca Corrente 2024 Ministero della Salute, both to GG. ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "All data generated or analysed during this study are included in this manuscript (and its supplementary information files). ",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "The authors declare that they have no conflict of interest. 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": "The authors declare that they have no conflict of interest. ",
"section_name": "Conflict of interest",
"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/fphar.2016.00099
|
Effect of Adoptive Transfer or Depletion of Regulatory T Cells on Triptolide-induced Liver Injury
|
The aim of this study is to clarify the role of regulatory T cell (Treg) in triptolide (TP)-induced hepatotoxicity.Female C57BL/6 mice received either adoptive transfer of Tregs or depletion of Tregs, then underwent TP administration and were sacrificed 24 h after TP administration. Liver injury was determined according to alanine transaminase (ALT) and aspartate transaminase (AST) levels in serum and histopathological change in liver tissue. Hepatic frequencies of Treg cells and the mRNA expression levels of transcription factor Forkhead box P3 and retinoid orphan nuclear receptor γt (RORγt), interleukin-10 (IL-10), suppressor of cytokine signaling (SOCS), and Notch/Notch ligand were investigated.During TP-induced liver injury, hepatic Treg and IL-10 decreased, while T helper 17 cells cell-transcription factor RORγt, SOCS and Notch signaling increased, accompanied with liver inflammation. Adoptive transfer of Tregs ameliorated the severity of TP-induced liver injury, accompanied with increased levels of hepatic Treg and IL-10. Adoptive transfer of Tregs remarkably inhibited the expression of RORγt, SOCS3, Notch1, and Notch3. On the contrary, depletion of Treg cells in TP-administered mice resulted in a notable increase of RORγt, SOCS1, SOCS3, and Notch3, while the Treg and IL-10 of liver decreased. Consistent with the exacerbation of liver injury, higher serum levels of ALT and AST were detected in Treg-depleted mice.These results showed that adoptive transfer or depletion of Tregs attenuated or aggravated TP-induced liver injury, suggesting that Tregs could play important roles in the progression of liver injury. SOCS proteins and Notch signaling affected Tregs, which may contribute to the pathogenesis of TP-induced hepatotoxicity.
|
[
{
"section_content": "Triptolide is isolated from the traditional Chinese medicine TWHF, which exhibits notable immune-regulative effects (Liu, 2011).TP has demonstrated a promising effect on the treatment of renal transplantation and autoimmune diseases in animal models by regulation of Tregs (Li et al., 2010;Zhou et al., 2011).Despite the benefits it provides, acute overdose or long-term administration of TP can cause severe liver injury and even death (Fu et al., 2011).The occurrence of drug-induced liver injury is a major problem in all phases of clinical drug development.In most cases, the mechanism of TP-induced hepatic injury remains unclear. Several reports have showed that Tregs play an important role in liver disorders (Sennello et al., 2005;Stross et al., 2012).Treg maintains immune homeostasis by suppressing excessive immune responses which otherwise would result in serious tissue damage (Kim et al., 2007).FoxP3 is characterized as a transcription factor required for Treg development (Yagi et al., 2004).IL-10 is produced by Tregs and other cell types.IL-10 is crucial for preventing exaggerated inflammation and thus protecting the host from immune-mediated damage (Sabat et al., 2010).The liver is an organ of complex immune responses and mainly provides protection by tolerating harmless self and foreign antigens (Doherty and O'Farrelly, 2000).When tolerance is broken, activated immune cells induce liver injury and hepatic inflammation by releasing proinflammatory cytokines and chemokines, which determines the extent of liver injury (Wang et al., 2015).Accumulating evidence suggests that anti-inflammatory Treg cells and proinflammatory Th17 cells have antagonistic effects on the progression of liver injury (Li et al., 2012).Therefore, it is critical to modulate the immune homeostasis during liver injury.Although the decrease of Tregs in liver injury has been reported (Wang et al., 2014a), the involvement of Tregs in noninfectious hepatic injury, such as TP-induced liver injury, has not been investigated.The purpose of this study is to identify the involvement of Treg cells in TP-induced liver injury. Suppressor of cytokine signaling proteins play a crucial role in preventing the cytokine responses and maintaining organ homeostasis.They are negative regulators of the JAK/STAT signaling pathway (Yoshimura et al., 2007).Eight SOCS proteins, SOCS1-SOCS7 and cytokine-inducible SH2-containing protein-1 (CIS-1) have been identified, of which SOCS1, 2, and 3 are the best characterized.Tregs are deficient in SOCS3 protein expression, which is needed for Treg to rapidly respond to cytokines to prevent unwarranted immune responses to selfantigens (Pillemer et al., 2007).The Notch signaling pathway control cell-fate decisions in T-cell development in the thymus and T-cell differentiation in the periphery (Deftos and Bevan, 2000).In mammals, there are four Notch receptors-Notch1-4 and five Notch ligands-Jagged-1 and -2 and Dll-1, -3, and -4 (Radtke et al., 2010).The Notch family contributes to induction of Treg cells and suppression of autoimmune diseases (Burghardt et al., 2014).Because SOCS and Notch signaling modulate differentiation and proliferation of T-helper (Th) cells.The second purpose of this study is to investigate the expression of SOCS and Notch/Notch ligands in TP-induced liver injury. Our previous investigations have reported that hepatic Treg and IL-10 decreased in TP-induced liver injury (Wang et al., 2014a), yet it has been unknown about the potential mechanisms regulating Tregs in TP-induced hepatotoxicity.In the present study, the percentage and phenotypic features of Tregs in the livers of mice were examined.The expression of SOCS proteins and Notch signaling were investigated.Either adoptive transfer or depletion of Tregs was conducted to investigate Tregs' roles in TP-induced liver injury.These results may lead to useful therapeutic approaches for TP-induced liver injury. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "Triptolide (purity >98%) was a gift from the Dermatological Disease Research Institute of the Chinese Academy of Medical Sciences (Nanjing, China).TP was reconstituted in propylene glycol and stored at -20 • C.Then, TP was freshly diluted to the appropriate concentrations with a 0.2% carboxymethylcellulose solution before use in the experiments. ",
"section_name": "Chemicals",
"section_num": null
},
{
"section_content": "Female C57BL/6 mice, 6-8 weeks of age, were purchased from the Vital River Experimental Animal Technology, Co., Ltd.(Beijing, China).All of the mice were housed under pathogen-free conditions and were provided with mouse chow and water ad libitum.The animals were maintained at a controlled temperature (22 • ± 2 • C) and photoperiod (12 h of light and 12 h of dark).The animals were acclimated to the laboratory for 1 week before the experiments.This study was approved by the Ethical Committee of China Pharmaceutical University, and Laboratory Animal Management Committee of Jiangsu Province (Approval No.: 2110748).The animal experiments were carried out in accordance with the approved guidelines. The mice were divided randomly into five groups (n = 12/group): (i) vehicle-treated control group, in which mice were given 0.2% carboxymethylcellulose solution (i.g.) at 24 h prior to sacrifice; (ii) TP group, in which mice were given TP (500 µg/kg, i.g.) at 24 h before sacrifice; (iii) adoptive transfer of Treg-pretreated TP group, in which mice were given CD4 + CD25 + Tregs (1 × 10 6 , i.v.) at 24 h before TP administration, then underwent TP administration and sacrifice at 24 h after TP administration; (iv) adoptive transfer of Teff-pretreated TP group, in which mice were given CD4 + CD25 -Teffs (1 × 10 6 , i.v.) at 24 h before TP administration, then underwent TP administration and sacrifice at 24 h after TP administration; (v) anti-CD25-pretreated TP group, in which mice were given purified anti-CD25 antibodies (200 µg, i.p., clone: PC61) at 48 h before TP administration (Tenorio et al., 2010), then underwent TP administration and sacrifice at 24 h after TP administration. ",
"section_name": "Animals and Treatment",
"section_num": null
},
{
"section_content": "Mouse CD4 + CD25 + Tregs and CD4 + CD25 -Teffs were isolated from the spleen with a MACS Treg isolation kit (Miltenyi Biotec, Auburn, CA, USA).The purity of the Treg cell was above 90%, as assessed by flow cytometry. ",
"section_name": "Cell Purification",
"section_num": null
},
{
"section_content": "The blood was collected in tubes without anticoagulant to obtain serum which was analyzed for the level of ALT and AST by using an automatic clinical analyzer (7080, HITACHI Ltd, Tokyo, Japan). ",
"section_name": "Blood Chemistry Aanalysis",
"section_num": null
},
{
"section_content": "Sections from the livers were removed and fixed in 10% neutralbuffered formalin.For the histopathological examination, all of the fixed organs were processed for embedding in paraffin, sectioned, and stained with H&E. ",
"section_name": "Histopathological Evaluations",
"section_num": null
},
{
"section_content": "Murine livers were passed through a 200-gage nylon mesh, and washed with cold PBS.The cell mixture was centrifuged at 50 × g for 2 min.The supernatant was then centrifuged at 800 × g for 10 min.For hepatic MNC isolation, the cell pellets were resuspended in 40% Percoll and centrifuged at 1250 × g for 15 min.Then the cell pellets were treated with lysis solution to remove erythrocytes and obtain hepatic MNCs.Next, cells were blocked with anti-CD16/32 (Becton Dickinson, San Diego, CA, USA) and labeled with anti-mouse CD4 antibodies (Becton Dickinson) before permeabilization with Cytoperm/Cytofix (Becton Dickinson) according to the manufacturer's instructions.After permeabilization, the cells were incubated with labeled antibodies that were specific for FoxP3 (Becton Dickinson).Then, the cells were centrifuged, and the pellets were washed to remove unbound antibodies.After surface and intracellular labeling, MNCs were evaluated by flow cytometry (Calibrate; Becton Dickinson, Palo Alto, CA, USA) and the data were analyzed using FlowJo version 10 software (FlowJo, Ashland, OR, USA). ",
"section_name": "MNC Isolation and Labeling",
"section_num": null
},
{
"section_content": "RNA was isolated from the liver sections with TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA).CDNA synthesis was performed under manufacturer's instructions by using the RevertAid First Strand cDNA Synthesis kit (Thermo Scientific, Waltham, MA, USA).Real-time PCR was performed in a 20-µL system which contained 10 µL of 1× SYBR Green Master Mix (Vazyme Biotech, Nanjing, China), 2 µL of cDNA, 6 µL of RNase/DNase-free water and 500 nM of each primer.The thermal cycler conditions included holds for 30 s at 95 • C, followed by 40 cycles of 5 s at 95 • C and 10 s at 60 • C. A melting curve analysis was performed for each reaction with a 65-95 • C ramp.The threshold cycle at which the fluorescent signal reached an arbitrarily set threshold near the middle of the log-linear phase of the amplification for each reaction was calculated, and the relative quantity of mRNA were determined.The mRNA levels were normalized against the mRNA levels of the housekeeping gene GAPDH.The primer sequences used were shown in Table 1. ",
"section_name": "RNA Extraction and Real-time PCR",
"section_num": null
},
{
"section_content": "Serum was used to determine the concentrations of IL-10 by ELISA (RayBio, Norcross, GA, USA) in accordance with the manufacturer's protocol. ",
"section_name": "Detection of Cytokines by ELISA",
"section_num": null
},
{
"section_content": "The data were expressed as the mean ± SEM.The groups were evaluated using a one-way analysis of variance (ANOVA) and Turkey's t-test.P-values <0.05 were considered to be statistically significant. ",
"section_name": "Statistical Analysis",
"section_num": null
},
{
"section_content": "Forward primer (5 -3 ) Reverse primer (5 -3 ) ",
"section_name": "Gene",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "The results demonstrated that ALT and AST levels significantly increased 24 hours after TP administration (Figures 1A,B).Histopathological changes in livers after TP administration showed necrosis with inflammatory cell infiltrating in hepatocytes (Figure 1C).However, adoptive transfer of Treg cells exhibited significantly lower levels of ALT and AST (Figures 1A,B) and ameliorated necrosis and inflammatory cell infiltrating in the liver (Figure 1C).Furthermore, adoptive transfer of Teffs reduced the TP-induced increase of AST, but didn't display more obvious alleviation of TP-induced liver injury.These findings demonstrated that adoptive transfer of Treg cells attenuated TP-induced liver injury accompanied with decreasing infiltration of inflammatory cells in the liver. ",
"section_name": "Adoptive Transfer of Tregs Ameliorates TP-induced Liver Injury",
"section_num": null
},
{
"section_content": "Administration of anti-CD25 antibodies and TP resulted in significantly higher ALT and AST levels in serum compared with the control group (Figures 2A,B).Moreover, H&E staining demonstrated severer hepatocyte necrosis and more inflammatory cell infiltration, compared with mice administered TP alone (Figure 2C).Mice administered the anti-CD25 antibodies alone had no obvious histopathological changes in the liver (Figure 2C). ",
"section_name": "Depletion of Treg Aggravates TP-induced Liver Injury",
"section_num": null
},
{
"section_content": "Mice treated with TP had significantly lower frequencies of hepatic CD4 + FoxP3 + cells (Figures 3A,B,E).Compared with the TP-administration group, adoptive transfer of Treg cells notably increased the frequencies of Treg cells in the liver (Figures 3D,E), yet adoptive transfer of Teffs didn't alter the percentage of hepatic CD4 + FoxP3 + cells (Figures 3C,E).Hepatic mRNA expressions of FoxP3, which is required for Treg development, showed tendencies to decrease in TP-administered mice (Figure 4A).Th17 cells, another subset of CD4 + T lymphocytes, were found to increase in TP-induced liver injury (Wang et al., 2014b).The hepatic expressions of ROR-γt, which is essential for Th17 cells differentiation, significantly increased after TP administration (Figure 4B).Thus, adoptive transfer of Treg not only significantly increased the hepatic mRNA expressions of FoxP3, but also remarkably reduced the mRNA levels of RORγt (Figures 4A,B).Adoptive transfer of Teffs didn't change the mRNA expression levels induced by TP.These findings indicated that adoptive transfer of Treg contributed to regulation of the Treg/Th17 balance in favor of Treg cells dominance, which attenuated Th17 response and established a tolerance environment in TP-induced liver injury. ",
"section_name": "Adoptive Transfer of Tregs Modulates the Treg/Th17 Balance in Favor of Treg Cells Dominance",
"section_num": null
},
{
"section_content": "The percentage of Treg cells significantly decreased in the murine livers of TP and anti-CD25 co-administered mice compared with the TP-administrated mice (Figure 5).Hepatic mRNA expression levels of FoxP3 significantly reduced, while ROR-γt noticeably increased in TP and anti-CD25 mAb co-administered mice (Figures 6A,B).These results indicated that depletion of Treg resulted in failure to modulate the Treg/Th17 balance and enhancement of Th17 cell expression. ",
"section_name": "Depletion of Treg Fails to Modulate the Treg/Th17 Balance and Increases Th17 Cells Expression",
"section_num": null
},
{
"section_content": "Interleukin-10, mainly produced by Treg cells, plays an important role in immune tolerance and anti-inflammation (Sabat et al., 2010).The results showed the mRNA levels of IL-10 in TP-treated livers significantly decreased.Adoptive transfer of Treg significantly increased the hepatic mRNA expressions of IL-10 (Figure 4C), but adoptive transfer of Teffs didn't change the mRNA expression levels of IL-10 induce by TP.The serum IL-10 levels remarkably decreased 24 h after TP administration.Tregspretreated mice had higher levels of IL-10, although it didn't show significant difference (Figure 4D).In addition, the serum IL-10 levels in Teffs-treated mice didn't significantly change compared with TP group.These results indicated that hepatic IL-10 contributed to Treg-mediated suppression of immune response in TP-induced liver injury. ",
"section_name": "Adoptive Transfer of Treg Cells Enhance IL-10 Secretion in Liver Tissue",
"section_num": null
},
{
"section_content": "Regulatory T-cell are likely to exhibit suppressive activity via secreting inhibitory cytokines IL-10 (Shevach, 2009).Our data showed not only hepatic mRNA expression levels of IL-10, but also serum IL-10 levels significantly reduced in TP and anti-CD25 mAb administered mice compared with the control group (Figures 6C,D). ",
"section_name": "Depletion of Treg Diminishes IL-10 Secretion in Liver and Serum",
"section_num": null
},
{
"section_content": "Suppressor of cytokine signaling is a negative regulator of STAT pathway and can be significantly induced under pathophysiology conditions in the liver (Yoshimura et al., 2007).The Notch signaling pathway plays critical roles in T-cell development, differentiation and liver homeostasis maintenance (Deftos and Bevan, 2000).Hepatic mRNA levels of SOCS3, Notch1, Notch3, Notch4, Jagged-1, Jagged-2, and Dll 4 significantly increased after TP administration.Adoptive transfer of Tregs remarkably inhibited the up-regulation of SOCS3, Notch1, and Notch3.Moreover, Teffs didn't change the gene expression levels induced by TP (Figure 7).These results suggested that SOCS3, Notch1 and Notch 3 might affect Tregs, which might contribute to the pathogenesis of TP-induced hepatotoxicity. ",
"section_name": "Adoptive Transfer of Tregs Down-regulates the Expressions of SOCS and Notch Signaling in TP-induced Liver Injury",
"section_num": null
},
{
"section_content": "Treg depletion further increased the gene expressions of SOCS1, SOCS3, and Notch3 compared with TP-administered mice (Figure 8).These results suggested that SOCS1, SOCS3, and Notch 3 might modulate Tregs in the development of TP-induced hepatotoxicity. ",
"section_name": "Depletion of Treg Up-regulates the Expressions of SOCS and Notch Signaling in TP-induced Liver Injury",
"section_num": null
},
{
"section_content": "Regulatory T cell can diminish potentially harmful immune responses, including inhibiting T-cell proliferation and blockading inflammatory cytokines release (Stockinger et al., 2001).Tregs are engaged in a variety of liver disorders, such as non-alcoholic fatty liver disease (Ma et al., 2007), ConAinduced liver injury (Erhardt et al., 2007) and biliary atresia (Liu et al., 2015).Tregs have reciprocal relationships with pro-inflammatory Th17 cells in the liver (Wang et al., 2014a).Th17 cell is a new lineage of CD4 + T cell subset, which induces the immune activation and is relevant to severity of liver injury (Apostolidis et al., 2011).In the present study, adoptive transfer of Treg not only significantly increased the hepatic mRNA expressions of FoxP3, but also remarkably reduced the mRNA levels of ROR-γt.These findings indicated that adoptive transfer of Treg contributed to modulating the Treg/Th17 balance in favor of Treg cells dominance, which attenuated Th17 response and established a tolerance environment in TP-induced liver injury.In contrast, depletion of Treg noticeably decreased the hepatic mRNA levels of FoxP3 and increased the ROR-γt expressions.Moreover, depletion of Treg resulted in immune activation and aggravation of TP-induced hepatotoxicity.Thus, Treg cells played important roles in maintaining homeostasis by suppressing excessive immune response which otherwise would result in serious liver injury. Interleukin-10 is mainly produced by Treg cells to exert effects on immune tolerance and anti-inflammation (Shevach, 2009).The mRNA levels of IL-10 in TP-treated livers significantly decreased, whereas adoptive transfer of Treg significantly increased the expressions of IL-10 in liver tissues.Moreover, depletion of Treg cell caused reduction of hepatic IL-10 expression levels and serum IL-10 levels.These observations indicated that IL-10, mainly produced and regulated by Tregs, involved in TP-induced liver injury model. Level of certain enzyme, such as ALT and AST, in serum can be monitored as a direct indicator of hepatocyte death and liver injury.Adoptive transfer of Treg cells significantly exhibited lower levels of ALT and AST in serum and attenuated inflammatory cell infiltrating in liver tissues.However, depletion of CD25 + cells showed aggravation of hepatotoxicity.From these lines of evidence, explaining the immunological and biological roles of hepatic Treg cells may contribute to providing a potential strategy for managing TP-induced liver injury. Suppressor of cytokine signaling proteins are a family of intracellular proteins that control cytokine signaling by suppressing cytokine signal transduction process (Yoshimura et al., 2007).The SOCS proteins, especially SOCS1 and SOCS3, play an essential role in mediating inflammatory responses in both immune cells and metabolic organs, such as liver (Galic et al., 2014).Conditional deletion of the SOCS3 gene revealed a role for SOCS3 as a negative regulator of the Treg subset with increased IL-10 and TGFβ production (Fitzgerald et al., 2009).In the present study, SOCS3 expression significantly increased in the liver after TP administration, which may be associated with hepatic Treg reduction.Whereas adoptive transfer of Tregs remarkably inhibited the expression of SOCS3, which may contribute to Treg expansion in the liver.In addition, depletion of Treg cells further increased the mRNA levels of SOCS1 and SOCS3 accompanied with more hepatic Treg reduction.The findings indicate that SOCS proteins are target genes for immune suppression induced by Treg cells. The Notch signaling pathway is engaged in T cell development and differentiation (Deftos and Bevan, 2000).In the liver, the Notch pathway is indispensable for liver homeostasis (Morell et al., 2013).FoxP3 has been demonstrated to be a downstream target of Notch signaling in human cells and Notch1 signaling is involved in Treg cell differentiation (Del Papa et al., 2013).TP administration induced significant increase of hepatic mRNA levels of Notch1, Notch3, Notch4, Jagged-1, Jagged-2, and Dll 4. Adoptive transfer of Tregs significantly inhibited the upregulation of Notch1 and Notch3, whereas Treg deficiency further increased the gene expressions of Notch3.Overall, these results revealed that Notch signaling contributed to the changes of adoptive transfer or depletion Treg in TP-induced liver injury. In summary, the present study clearly demonstrated a potent protective role of Tregs in TP-induced liver injury and this protection may be mediated by enhancing the secretion of IL-10.In addition, SOCS proteins and Notch signaling contributed to the changes of adoptive transfer or depletion of Treg in TP-induced liver injury.These findings may place Treg cells as potential targets for pharmacotherapy, though the detailed investigations on mechanism need to be carried out in the future. ",
"section_name": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "We thank Professor Chen Yun for providing triptolide, who is from the Dermatological Disease Research Institute of the Chinese Academy of Medical Sciences (Nanjing, China). ",
"section_name": "ACKNOWLEDGMENT",
"section_num": null
},
{
"section_content": "XW, LZ, and ZJ designed the experiments.XW performed the experiments.XW and ZJ analyzed and discussed the data.XW, LS, LZ, and ZJ wrote the paper.All authors contributed to the editing of the paper and to scientific discussions. The present study was supported by grants from the Natural Science Foundation of Jiangsu Province (BK20151439) and the National Natural Science Foundation of China (No. 81573514, No. 81573690, No. 81173651, No. 81274146, No. 81102887, and No. 81320108029).The study was partially supported by the 111 Project (No. 111-2-07). 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": "XW, LZ, and ZJ designed the experiments.XW performed the experiments.XW and ZJ analyzed and discussed the data.XW, LS, LZ, and ZJ wrote the paper.All authors contributed to the editing of the paper and to scientific discussions. ",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "The present study was supported by grants from the Natural Science Foundation of Jiangsu Province (BK20151439) and the National Natural Science Foundation of China (No. 81573514, No. 81573690, No. 81173651, No. 81274146, No. 81102887, and No. 81320108029).The study was partially supported by the 111 Project (No. 111-2-07). ",
"section_name": "FUNDING",
"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
}
] |
10.1186/1471-2407-7-152
|
The BRCA1/2 pathway prevents hematologic cancers in addition to breast and ovarian cancers
|
The present study was designed to test the hypothesis that inactivation of virtually any component within the pathway containing the BRCA1 and BRCA2 proteins would increase the risks for lymphomas and leukemias. In people who do not have BRCA1 or BRCA2 gene mutations, the encoded proteins prevent breast/ovarian cancer. However BRCA1 and BRCA2 proteins have multiple functions including participating in a pathway that mediates repair of DNA double strand breaks by error-free methods. Inactivation of BRCA1, BRCA2 or any other critical protein within this "BRCA pathway" due to a gene mutation should inactivate this error-free repair process. DNA fragments produced by double strand breaks are then left to non-specific processes that rejoin them without regard for preserving normal gene regulation or function, so rearrangements of DNA segments are more likely. These kinds of rearrangements are typically associated with some lymphomas and leukemias.Literature searches produced about 2500 epidemiology and basic science articles related to the BRCA pathway. These articles were reviewed and copied to a database to facilitate access. Meta-analyses of statistical information compared risks for hematologic cancers vs. mutations for the components in a model pathway containing BRCA1/2 gene products.Deleterious mutations of genes encoding proteins virtually anywhere within the BRCA pathway increased risks up to nearly 2000 fold for certain leukemias and lymphomas. Cancers with large increases in risk included mantle cell lymphoma, acute myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, and prolymphocytic leukemia. Mantle cell lymphoma is defined by a characteristic rearrangement of DNA fragments interchanged between chromosomes 11 and 14. DNA translocations or rearrangements also occur in significant percentages of the other cancers.An important function of the BRCA pathway is to prevent a subgroup of human leukemias and lymphomas that may involve non-random, characteristic gene rearrangements. Here, the genetic defect in BRCA pathway deficiencies is a chromosomal misrepair syndrome that may facilitate this subgroup of somatic cancers. Inactivation of a single gene within the pathway can increase risks for multiple cancers and inactivation of a different gene in the same pathway may have similar effects. The results presented here may have clinical implications for surveillance and therapy.
|
[
{
"section_content": "BRCA1 and BRCA2 proteins are thought to be essential to prevent breast/ovarian cancer largely because of the high lifetime risks faced by carriers of mutations in the corresponding genes.More modest increases in risk for other cancers have also been noted [1][2][3][4][5].Basic science studies find multiple biologic functions for BRCA1 and BRCA2 proteins [6][7][8][9][10][11][12][13][14][15], including participating within a pathway that mediates error-free repair of DNA double strand breaks by homologous recombination [15]. Fig. 1 summarizes a model for this error-free double strand break repair pathway (based on reference [16]).BRCA1 and BRCA2 gene products are placed within a sequence encompassing the MRE11, Rad50 and NBS1 complex (MRN complex), ATM, CHEK2, BRCA1, BRCA2, and Fanconi anemia proteins.For the purposes of this paper, this model will be referred to as the \"BRCA pathway.\" A critical protein function lost from anywhere within this error-free repair BRCA pathway may force repair of DNA double strand breaks by lower fidelity, error prone methods.Risks for cancers mediated by such errors should then greatly increase.Lymphomas and leukemias can be associated with large gene rearrangements, which can be pictured as arbitrary rejoining of broken DNA fragments.For example, almost all mantle cell lymphomas have a characteristic interchange between pieces of chromosomes 11 and 14 [t(11;14)(q13;q32)].In some leukemias, the make-up of a fusion protein may bear witness to other abnormal repairs [e.g.[17]].Error-tolerant repair may also leave other signs such as in the acute myeloid leukemias, where there may be evidence of abnormal gene fusions, duplications, inversions, deletions or reciprocal translocations [18].The present study was designed to test the hypothesis that inactivation of a critical component of the BRCA pathway would favor gene rearrangements that underlie some lymphomas and leukemias.The results show that risks for a subset of leukemias and lymphomas increase up to nearly 2000 fold.The results may have clinical implications for surveillance and chemotherapy. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "The study was designed to review the risks for leukemias and lymphomas associated with a deleterious mutation within a prototype BRCA pathway (Fig. 1) for error-free double strand break repair.The purpose of this article is not to examine functionality of specific gene variants, but rather to examine the effect of loss of gene function anywhere within a testable pathway on risk for specific hematologic cancers.For many of the studies examined, especially case series, the exact genetic variant is unknown but loss of gene function (regardless of the reason) was confirmed by other means (e.g.RNA, protein or other Schematic model for the \"error-free\" BRCA double strand break repair pathway Brief overview of components within the BRCA pathway used here as a working model that was tested here Figure 1 Schematic model for the \"error-free\" BRCA double strand break repair pathway Brief overview of components within the BRCA pathway used here as a working model that was tested here.The model is based largely on reference 16.BRCA2 is the same as FANCD1 and the interaction between BRCA1 and Fanconi anemia protein J is shown.While the gene products shown represent the over-all pathway, \"errorfree\" double strand break repair by homologous recombination undoubtedly involves other proteins but the discussion is limited to those shown.Not shown are details of the 13 Fanconi anemia gene products and additional components including EMSY, a whole family of RAD51 related proteins, DCC, cohesins and accessory proteins.Deficiency states may be rare or unknown for these additional proteins and large epidemiologic studies are uncommon.Other protein kinases related to ATM carry out similar functions in response to other genotoxic stresses, and some of them collaborate with ATM.Proteins within the pathway also interact with other branches of the DNA damage response and with further proteins.PubMed, PubMed Central, Google, and Google scholar searches were conducted to collect relevant research articles related to the model BRCA pathway.These searches were for epidemiology studies published within the last ~20 years and basic science articles published within the past ~15 years.About 2500 articles were reviewed and copied to a database to facilitate search and further review. Where available statistical information permitted, metaanalysis with heterogeneity testing was conducted comparing cancer risk vs. deleterious mutation of a pathway gene. Case-control, cohort and prevalence studies were reviewed.Data from studies that measured cancer incidences associated with epigenetic modification of pathway genes and/or alterations in protein or mRNA levels were also included.Epidemiologic studies were excluded in whole or in part if they did not provide required data or permit calculation of required information or if they were superseded or subsequently invalidated.The rarity of mutations in some molecules limited data available and limited the analysis of some BRCA pathway components. As far as possible, statistical analyses were limited to gene variants either known to eradicate normal protein function or to severely lower normal levels.All the mutations were spontaneously occurring and/or inherited except for therapy related (somatic) inactivation of BRCA1 in acute myeloid leukemia (AML).To verify that therapy related disease did not bias the results it was compared to data for primary AML. Epidemiologic data was tabulated as odds ratios or relative risks: for ATM associations with NHL as MCL, with ALL, CLL, and PLL; for Fanconi anemia gene associations (13 known genes) with primary AML, with leukemia before age 15 and with ALL; for BRCA1 associations with primary and therapy related AML and with CML; for BRCA2 associations with AML, ALL and CLL; for NBS1 associations with lymphomas, ALL and NHL; and for CHEK 2 associations with CLL. The DerSimonian-Laird random effects model [21] was used throughout since it relaxes the assumption of a common effect due to mutation.This may be more appropriate here than fixed effects models since inactivating mutations can in theory have different targets with different effect sizes.However, the uncertainty bounds for random effects are more conservative and often larger.When at least three studies were available, meta-analysis was performed.For the The prevalence of ATM mutation heterozygotes in the general population is widely cited as 0.3% to 1%.The incidence of biallelic mutations which are required to cause the hereditary disease A-T is much smaller (3/million to 11/million) [22,23].Use of any value within this range as a control would give much larger risks.However, a population prevalence of 1% for ATM mutations was used to prevent overstating differences from the general population. Heterogeneity was calculated as non-combinability of odds ratios by the Breslow-Day method, from the inconsistency statistic [24], by a moment based method and graphically from L'Abbe plots.None of the meta-analyses presented showed evidence for heterogeneity by these criteria.Chi-square tests on combined odds ratios were performed.Bias was assessed using the method of Egger and by inspecting funnel plots for asymmetry [25].There was no statistical evidence of publication bias for summary estimates (results not shown). A general limitation of meta-analysis is that access to original data is limited or the data is so old that some calculations in publications are impossible to reproduce.Fortunately, some articles used in meta-analyses contained both raw and final calculated data.This enabled control experiments to check the validity of calculations based on raw data.Testing raw instead of final data gave no or very small errors as confirmed dozens of times.To rule out computer program errors, the RevMan program from the Cochrane Review Group was used to verify some calculations made by StatsDirect.Microsoft Excel with the data-analysis add-in was used for some calculations. ",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "The present study was designed to test the hypothesis that inactivation of virtually any component within the BRCA pathway would increase risks for lymphomas and leukemias.Risks were summarized [see Additional file 1] for leukemias and lymphomas vs. mutations or aberrations at numerous steps within the model pathway in Figure 1. Each of the genes within the BRCA pathway is considered below.The conclusion emerges that inactivation of any of these genes greatly increases risks for a subgroup of leukemias and lymphomas.This subgroup includes (B-cell) mantle cell lymphoma (MCL), acute myeloid leukemia (AML), T-cell acute lymphocytic leukemia (T-ALL), chronic lymphocytic leukemia (B-CLL) and T-cell prolymphocytic leukemia (T-PLL).The large increases in risk suggest that preventing these diseases must be an important physiologic function of the complete BRCA pathway.The results also suggest a mechanism for this function. ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "9 studies of the incidence of ATM mutations in MCL from a total of 363 patient samples were summarized [see Additional file 1].Meta-analysis seemed appropriate initially because all the studies found very strong odds ratios for an ATM-MCL association, so all 9 studies have the same general pattern (criteria used by the Cochrane Review group). Combining the 9 studies then as described in Methods [21][22][23][24][25], gave 70.26 [95% CI = 34.59-142.72]as the minimum odds ratio that a mantle cell lymphoma contains an ATM mutation (Table 1).The chi 2 test value that the pooled odds ratio differs from 1 was 138.30,P < 0.0001.No significant heterogeneity was found by multiple criteria given in Methods but there are only 9 estimates based on 363 people, a comparatively small pooled population. Additional statistical confirmation that MCL is associated with ATM mutation was obtained for the data used.The product-moment linear correlation coefficient was calculated for total experimental samples with MCL vs. experimental samples with ATM mutation.The correlation coefficient value was 0.95 indicating a strong association. The translocation t(11;14)(q13;q32) is present in almost all mantle cell lymphomas [19,20].This translocation is consistent with the misrepair of a double strand break. The MCL tumors associated with this translocation corre- ",
"section_name": "Inactivation of the BRCA pathway gene ATM favors a translocation associated with mantle cell lymphoma",
"section_num": null
},
{
"section_content": "There are very high odds ratios for an association of an ATM deficit not only with MCL, but also with T-ALL, B-CLL, and T-PLL (Table 1).Substantial percentages of any of these diseases associate with misrepair of some double strand break leading to gene rearrangement or deletion within an affected hematopoietic cell lineage.Fusion proteins and/or gene rearrangements have been documented in about 30% of 2367 children with ALL [26], in 11% of B-CLL [27] and in high percentages of atypical CLL with poor prognosis [28].Table 1 and Additional file 1 also show that ALL and CLL can be associated with any of several BRCA pathway malfunctions.100 fold higher than in the general population.In A-T there is a recurrent malignancy similar to T-PLL with a similar course, a similar immunophenotype, and similar cytogenetics [reference [32] and Table 2].T-PLL occurs at high frequency in A-T families compared to non A-T families [33].These considerations support the association between ATM mutations and T-PLL. In some cases, there is no truly reliable way to distinguish somatic from inherited mutations in the BRCA pathway. It is unlikely that this biases the results.The first row in Table 2 gives the risk for some leukemias and lymphomas in A-T patients.Risks for A-T patients are similar to those summarized in Table 1 for ATM mutations in people who do not have hereditary A-T [also see Additional file 1].Somatic BRCA1 data can also be compared to that for the hereditary disease Fanconi anemia because BRCA1 interacts with the Fanconi protein FANCJ.Therapy related inactivation of BRCA1 (preventing its interaction with FANCJ) causes risks for AML comparable to risks for AML due to hereditary Fanconi anemia (see below). ",
"section_name": "ATM mutation: evidence for association with the leukemias T-ALL, B-CLL, and T-PLL",
"section_num": null
},
{
"section_content": "I also examined potential associations between hereditary Fanconi anemia gene mutations and risk of hematologic cancers.Results were available from about 80 years of published data in Fanconi anemia databases [34][35][36].Data exists from a relatively large number of patients in 3 summary studies.The Fanconi anemia studies each report very large hazard ratios for early leukemias and calculated relative risks are also high [see Additional file 1].Fanconi anemia patients have a relative risk of 703.35 for AML as calculated by meta-analysis (Table 1).Frequent spontaneous chromosome breaks and gross-chromosomal rearrangements are visible in Fanconi anemia cells (Table 2), consistent with large increases in risk for cancers mediated by chromosome rearrangements.Some data predates the ability to identify individual Fanconi mutations.This merged data was justified for use here because of the close interactions and relationships among Fanconi proteins (Figure 1). ",
"section_name": "Fanconi Anemia genes within the BRCA pathway and early leukemias",
"section_num": null
},
{
"section_content": "Independent and unrelated lines of investigation strongly implicate BRCA1 and BRCA2 deficiencies in hematologic cancers.This information is summarized below and there is additional corroborating evidence [15,[37][38][39][40][41][42][43][44] Relationships and critical interactions exist among BRCA1, BRCA2 and Fanconi anemia proteins(e.g.Fig. 1 and Table 2).Because of connections between Fanconi proteins and leukemias, these relationships further implicate BRCA1/BRCA2 deficits in leukemias.As an example of interactions between Fanconi anemia proteins and BRCA1/2 proteins, the Fanconi anemia protein FANCJ forms an essential complex with BRCA1.This complex brings FANCJ (together with replication protein A) into nuclear foci at the site of DNA damage.FANCJ then unwinds DNA sufficiently so that error-free repair can begin (Table 2).In kindreds who have BRCA2 (FANCD1) mutations on top of another Fanconi anemia mutation, leukemia occurs at a median age of 2.2 years instead of 13.4 years [reference [37] and Additional file 1]. Some epidemiologic studies show increased risks for leukemia/lymphoma [1] in identified BRCA1 or BRCA2 mutation carriers [46,47] and in large populations eligible for mutation testing [48][49][50][51][52][53].Family history can be used to determine eligibility for mutation testing and can estimate the likelihood that a BRCA1 or BRCA2 mutation exists within the family [1].Rauscher et al [54] reported that family history of breast cancer increased risk due to a range of leukemia risk factors that were generally weak or non-existent when considered alone.Combined with a family history of breast cancer, ever-smoking [RR 11 = 2.4, CI = 1.2-4.8],general solvent exposure (RR 11 = 1.9, CI = 1.1-3.4),aromatic hydrocarbon exposure (RR 11 = 3.8, CI = 1.1-14), and diagnostic ionizing radiation exposure (RR 11 = 2.1, CI = 1.2-3.8)were all associated with increased leukemia incidence.There was no increased incidence associated with any of these exposures in the absence of a family history of breast cancer [54]. ",
"section_name": "BRCA1 and BRCA2 genes in the BRCA pathway and AML, leukemias and lymphomas",
"section_num": null
},
{
"section_content": "Abnormalities in a complex containing the BRCA pathway proteins MRE11, Rad50 and NBS1 (MRN complex) also associate with leukemia and lymphoma.Rarely, hypomorphic mutations in MRE11 occur in individuals with \"Ataxia-Telangiectasia-Like-Disorder\" (ATLD).Lymphocytes from ATLD patients may carry chromosome translocations identical to lymphocytes from A-T patients [55], implying a corresponding predisposition to leukemias and lymphomas ",
"section_name": "MRE11-Rad50-NBS1: a complex of BRCA pathway genes and lymphomas and leukemias",
"section_num": null
},
{
"section_content": "Checkpoint kinase 2 (CHEK2) participates in the BRCA pathway by phosphorylating BRCA1, promoting cell cycle arrest, and activating DNA repair in genetically damaged cells [57].CHEK2 may also affect risks for hematologic cancers.The CHEK2 variant I157T is significantly associated with CLL [see Additional file 1].CHEK2 mutations were uncommon in NHL but 9% of these tumors showed either total or near-total absence of the CHEK2 protein [58].CHEK2 alterations responsible for these low levels occur in a subset of aggressive lymphomas having a relatively high number of chromosomal imbalances [58].CHEK2 may also have some relationship to promyelocytic leukemia (PML) because CHEK2 phosphorylates the tumor suppressor PML gene protein leading to apoptosis [59] Although the composition of nuclear bodies containing PML varies during the cell cycle, they may also contain other members of the BRCA pathway and participate in double strand break repair [60,61]. ",
"section_name": "CHEK2: a BRCA pathway gene and lymphomas and leukemias",
"section_num": null
},
{
"section_content": "Participation of BRCA pathway deficiencies in leukemias and lymphomas can be explained by incorporating features of overlapping theories for how cancers arise.These theories are differentiation-maturation mutations cooperating with proliferation/survival mutations, lineageaddiction or -dependency models, and the existence of \"cancer stem cells\" arising from an ordered sequence of phenotypically distinct stem-cell and intermediate-precursor populations [62][63][64].Applying elements of these current theories helps clarify the present results as discussed below. Some lymphomas and leukemias are defined by non-random, characteristic gene rearrangements [e.g.[65][66][67][68]] but people can have small numbers of cells containing one of these translocations that may not progress to cancer.Thus additional abnormalities are required to create cancer.According to one hypothesis [62], two kinds of gene rearrangements or other mutations cooperate to produce leukemias and lymphomas (i-ii).i. Gene rearrangements or other mutations that give a growth and/or survival advantage to malignant cells.ii.Gene rearrangements or other mutations that impair differentiation. Mantle cell lymphoma (MCL) is associated here with a BRCA pathway deficit and this association is consistent with current cancer models.In almost all cases of MCL, there is a characteristic exchange of fragments between chromosome 11 and chromosome 14.The rearrangement occurs within a subset of naive pregerminal center cells in the B-cell lineage.This \"MCL translocation\" results in the juxtaposition of the BCL1 gene (cyclin D1) and the immunoglobulin heavy chain locus.The MCL translocation causes cyclin D1 to become overexpressed because it comes under the control of the highly active immunoglobulin gene enhancer.Overexpressed cyclin D1 then probably functions as an oncogene by accelerating cell division.This gives a growth advantage to cells containing the rearrangement.In some systems, cancer cell lines are dependent on a cyclin D1 oncogene for survival (oncogene addiction).Abnormal cells here with the cyclin D1 oncogene, have a growth advantage that makes further mutations more likely to accumulate.A BRCA pathway deficit causes an underlying deficiency in error-free repair that increases the number of abnormal cells and adds further to the chances for additional abnormalities.However, the aberrant B-cell lineage may well condition the range of mutations allowed because of embedded differentiation or developmental programs.\"Atypical CLL\" shares cytogenetic and immunologic features with MCL [32,69].The same \"MCL translocation\" between chromosomes 11 and 14 occurs in \"atypical CLL.\" [69], consistent with this macro-genomic alteration being restricted to the B-cell lineage. AML is another example of a disease associated with a deficit in BRCA-pathway-mediated DNA repair.The hallmark of all AML types is a severe block in myeloid differentiation.In previous sections of this paper, Fanconi anemia patients were shown to have >700 fold increase in combined relative risk for AML (Table 1).In Fanconi anemia, the BRCA pathway deficiency leads to visibly increased numbers of chromosome breaks, gaps, rearrangements, and quadriradii in the presence of DNA damaging agents.This may result from a documented increase in repair by the less specific process of non-homologous end joining. AML is the generic term for a group of myeloid leukemias that have a clonal expansion of immature myeloid progenitor stages (blasts) in the bone marrow, blood or other tissues.Different categories of AML can depend on a particular mutation event that creates a block in differentiation and the stage within the myeloid lineage when the event occurs.Translocation events, duplications, inversions, or deletions would be favored by BRCA pathway defects and they represent potential ways to create a differentiation block typical of AML.Although other types of mutation also create differentiation blocks in AML, the large increases in relative risk in Fanconi anemia suggests that gene rearrangements are important.In Fanconi anemia, translocations occur at a rate that is at least ten times greater than normal after exposure to ionizing radiation [70]. An example of a translocation capable of creating a differentiation block is the recurring t(3;12)(q26;p13) translocation.In a Fanconi anemia patient, this rearrangement was present in the bone marrow at the time of initial diagnosis of myelodysplastic syndrome (often a precursor of AML).The patient had a normal constitutional karyotype but AML then developed.When acute transformation to AML occurred, cytogenetic analysis found multiple chromosome deletions and rearrangements typical of Fanconi anemia [71].Fanconi anemia is a rare inherited disease, but the same t(3;12) translocation is sometimes the first and the only cytogenetic abnormality found in AML patients who do not have hereditary Fanconi anemia.This particular rearrangement is thought to predispose to AML as follows [72].It causes overexpression of the EVI-l gene because EVI-1 becomes driven by the TEL promoter [71,62].Normally EVI-1 is expressed in early myeloid progenitor cells where it helps determine whether progenitors differentiate or proliferate.Abnormal EVI-1 expression probably contributes to AML by interfering with other genes controlling the commitment to differentiate.These progenitors are designed to proliferate rapidly and then to differentiate.Failure to induce timely differentiation might result in a prolonged proliferation phase favoring the accumulation of additional cooperating events.This places the progenitors at much higher risk for leukemia [63].A background of hereditary Fanconi anemia would greatly increase chances for gene rearrangements and deletions in progenitors both as initial and as cooperating events. A variety of gene rearrangements due to misrepaired double strand breaks also occur frequently in other diseases associated here with BRCA pathway deficiencies.In some cases of T-PLL, one gene rearrangement deregulates the expression of the T-cell receptor.Similarly, any of several recurring chromosomal translocations can be detected in substantial numbers of cases of childhood ALL. Defects in the BRCA pathway increase the risks for a subset of lymphomas and leukemias that are probably associated with gene rearrangements.However a BRCA pathway deficit does not cause the underlying gene rearrangements.The deficit allows more mistakes in double strand break repair, increases the numbers of cells with mistakes and then permits abnormal cells to survive. In myeloid leukemias, certain sites may associate with up to 40 different gene partners and chromatin structural elements closely associate with such breakpoints [68].Some of these translocations have prognostic significance.Perhaps certain chromosome regions are selected for these rearrangements because they are more actively transcribed and exposed in a transcription complex [73].The proximity between neighboring chromosomes may also be an influence. Some tumors [see Additional file 1] contain evidence that ATM deficiency compromises the BRCA pathway regardless of other pathways involving ATM.Gene fusions or other rearrangements often found in some of these tumors bear witness to a double strand break repaired by error-prone methods.The cancers reported here are thus primarily somatic in origin but the predisposition to misrepair of DNA breaks and chromosomal instability may be inherited.Inactivation of a single gene can increase risks for multiple cancers and inactivation of a different gene in the same pathway may have similar effects [see Additional file 1]. The deficiencies that increase risk for leukemias and lymphomas may well be helpful in understanding other cancers in BRCA1/2 mutation carriers.The involvement of BRCA pathway deficits in a subset of hematologic cancers has implications for surveillance and for therapy in hematologic and perhaps in other cancers.These deficits suggest the need for improved surveillance.They also present a vulnerability that may be exploited during therapy. Reciprocal translocations and other chromosome rearrangements also occur in breast and in ovarian tumors [74,75].Comparative genome hybridization has shown that human epithelial breast tumors undergo widespread gains and losses of chromosomes early in their development, correlating well with the presence of complex chromosomal rearrangements [76].In comparing hematological and epithelial cancers in 2001, Ponder asked \"Are there similar mechanisms among the more complex chromosomal changes in epithelial malignancies, or do epithelial cancers have different genetic mechanisms of development?\"[77].The data in the present paper adds the information that the same BRCA pathway can be disabled in both breast and hematological cancers, showing that further consideration of Ponder's question may be very helpful. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "BRCA1 and BRCA2 are critical to prevent breast and ovarian cancers in mutation carriers but the proteins participate in processes that are fundamental for survival in other types of cells.The genetic defect accompanying BRCA pathway deficiencies studied here is a chromosomal misrepair syndrome.This work shows that the pathway containing BRCA1/2 gene products is essential to prevent a group of leukemias and lymphomas.The results may have clinical implications for surveillance and chemotherapy in these and perhaps in other cancers. Publish with Bio Med Central and every scientist can read your work free of charge ",
"section_name": "Conclusion",
"section_num": null
}
] |
[
{
"section_content": "I am very grateful and wish to extend special thanks to Dr. Jack Kaplan and the UIC College of Medicine for their general support.Dr. E.P. Cohen assisted with an early helpful review.Dr. Jan Lubinski contributed invaluable critical review of the completed manuscript.Dr. Harvey Friedenson was of great help in editing the final draft.Dr. Denys Wheatley donated his valuable editorial services through Biomedes.It is a pleasure to thank biostatistician Weihua Gao, MS in the Quantitative Biomedical Sciences Program and the Quantitative Biostatistics Core of the UIC Cancer Center, for helpful discussions and review of the statistical methodology and data. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "AML, acute myeloid leukemia; A-T, ataxia-telangiectasia; ATLD, Ataxia-Telangiectasia-Like-Disorder; ATM, ataxiatelangiectasia mutated; B-CLL, (B-cell) chronic lymphocytic leukemia; CHEK2, Checkpoint kinase 2; CI = 95% Confidence Interval; CML, chronic myelogenous leukemia; MCL, mantle cell lymphoma; MRN complex, complex of MRE11, Rad50 and NBS1; NBS, Nijmegan breakage syndrome; NHL, non-Hodgkins lymphoma; OR, odds ratio; PML, promyelocytic leukemia; RR, relative risk; T-ALL, T-cell acute lymphocytic leukemia; T-PLL, Tcell prolymphocytic leukemia. The author(s) declare that they have no competing interests. The author was the sole contributor. Mutation of BRCA pathway components in leukemias and lymphomas.Summaries of case-control, cohort, and basic science research studies that provided numerical, statistical and/or patient data for BRCA pathway gene deficits vs. leukemias and lymphomas.Click here for file [http://www.biomedcentral.com/content/supplementary/1471-2407-7-152-S1.doc] The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2407/7/152/prepub ",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "AML, acute myeloid leukemia; A-T, ataxia-telangiectasia; ATLD, Ataxia-Telangiectasia-Like-Disorder; ATM, ataxiatelangiectasia mutated; B-CLL, (B-cell) chronic lymphocytic leukemia; CHEK2, Checkpoint kinase 2; CI = 95% Confidence Interval; CML, chronic myelogenous leukemia; MCL, mantle cell lymphoma; MRN complex, complex of MRE11, Rad50 and NBS1; NBS, Nijmegan breakage syndrome; NHL, non-Hodgkins lymphoma; OR, odds ratio; PML, promyelocytic leukemia; RR, relative risk; T-ALL, T-cell acute lymphocytic leukemia; T-PLL, Tcell prolymphocytic leukemia. ",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "The author(s) declare that they have no competing interests. ",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "The author was the sole contributor. ",
"section_name": "Authors' contributions",
"section_num": null
},
{
"section_content": "",
"section_name": "Additional material",
"section_num": null
},
{
"section_content": "Mutation of BRCA pathway components in leukemias and lymphomas.Summaries of case-control, cohort, and basic science research studies that provided numerical, statistical and/or patient data for BRCA pathway gene deficits vs. leukemias and lymphomas.Click here for file [http://www.biomedcentral.com/content/supplementary/1471-2407-7-152-S1.doc] ",
"section_name": "Additional file 1",
"section_num": null
},
{
"section_content": "The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2407/7/152/prepub ",
"section_name": "Pre-publication history",
"section_num": null
}
] |
10.1186/s13027-022-00423-5
|
Mammalian tumor-like organs. 2. Mammalian adipose has many tumor features and obesity is a tumor-like process
|
<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>In previous publications, the author developed the theory of<jats:italic>carcino-evo-devo</jats:italic>, which predicts that evolutionarily novel organs should recapitulate some features of tumors in their development.</jats:p></jats:sec><jats:sec><jats:title>Main text</jats:title><jats:p>Mammalian adipose is currently recognized as a multi-depot metabolic and endocrine organ consisting of several adipose tissues. Although lipid-storing cells and proteins are ancient, the adipose organ as a whole is evolutionarily novel to mammals. The adipose expansion has remarkable similarities with the growth of solid tumors. These similarities are the following: (1) The capability to unlimited expansion; (2) Reversible plasticity; (3) Induction of angiogenesis; (4) Chronic inflammation; (5) Remodeling and disfunction; (6) Systemic influence on the organism; (7) Hormone production; (8) Production of miRNAs that influence other tissues; (9) Immunosuppression; (10) DNA damage and resistance to apoptosis; (11) Destructive infiltration in other organs and tissues. These similarities include the majority of “hallmarks of cancer”. In addition, lipomas are the most frequent soft tissue tumors, and similar drugs may be used for the treatment of obesity and cancer by preventing infiltration. This raises the possibility that obesity, at least in part, may represent an oncological problem. The existing similarities between adipose and tumors suggest the possible evolutionary origin of mammalian adipose from some ancestral benign mesenchymal hereditary tumors. Indeed, using a transgenic inducible zebrafish tumor model, we described many genes, which originated in fish and were expressed in fish tumors. Their human orthologs<jats:italic>LEP</jats:italic>,<jats:italic>NOTCH1</jats:italic>,<jats:italic>SPRY1</jats:italic>,<jats:italic>PPARG</jats:italic>,<jats:italic>ID2</jats:italic>, and<jats:italic>CIDEA</jats:italic>acquired functions connected with the adipose organ. They are also involved in tumor development in humans.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>If the hypothesis of the evolutionary origin of the adipose organ from the ancestral hereditary tumor is correct, it may open new opportunities to resolve the oncological problem and the problem of the obesity epidemic. New interventions targeting<jats:italic>LEP</jats:italic>,<jats:italic>NOTCH1</jats:italic>,<jats:italic>SPRY1</jats:italic>,<jats:italic>PPARG</jats:italic>,<jats:italic>ID2</jats:italic>, and<jats:italic>CIDEA</jats:italic>gene network, in addition to what already is going on, can be designed for treatment and prevention of both obesity and tumors.</jats:p></jats:sec>
|
[
{
"section_content": "In previous publications, the author developed the theory of carcino-evo-devo, which describes the coevolution of normal and neoplastic development [1,2].I formulated the hypothesis of evolution by tumor neofunctionalization (below I will call it \"the main hypothesis\"), which suggested that the evolutionary role of hereditary tumors might consist in supplying evolving multicellular organisms with extra cells masses for expression of evolutionarily novel genes and the origin of new cell types, tissues, and organs [1,2]. Several non-trivial predictions of the main hypothesis have been confirmed in my laboratory ( [1][2][3][4], reviewed in [5]).One non-trivial prediction of the main hypothesis is that evolutionarily novel organs if they indeed originated from hereditary tumors or tumor-like structures, should recapitulate some features of tumors in their development.That is why in previous articles [2,3,5] I was looking for the data that might confirm this prediction in the literature, and also performed some experiments in my lab.The first paper in this series [5] reviewed the evidence that evolutionarily novel organs such as the placenta, mammary gland, prostate, and infantile brain indeed have many features of tumors including the regulated invasiveness at certain stages of their development and higher incidence of cancer.In that paper, I suggested calling evolutionarily new normal organs, which have many tumor features, the tumor-like organs for brevity [5]. In the present paper, the author reviews the evidence that mammalian adipose, the other evolutionarily novel organ of mammals, has many tumor features as well, and obesity is a tumor-like process.A hypothesis of the possible evolutionary origin of mammalian adipose from hereditary mesenchymal tumors is formulated and supporting data obtained in the author's lab are discussed. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Main text",
"section_num": null
},
{
"section_content": "Adipose is a metabolic and endocrine organ operating \"as a structured whole\" [6,7].The concept of adipose as a large multi-depot organ with discrete anatomy was developed by S. Cinti [6,[8][9][10].The adipose organ consists of white adipose tissue and brown adipose tissue distributed in a series of subcutaneous and visceral depots.Each depot of the organ has its own vascular and nerve supply.White adipose tissue (WAT) is involved in triglyceride/energy storage, and brown adipose tissue (BAT)-in energy expenditure.WAT and BAT differ in morphology and location [10].The third type of fat, beige adipose tissue, resembles brown adipose morphologically and functionally, but its development is closer to the development of white adipose tissue [10,11].Some authors also consider bone marrow adipose as a separate type of adipose tissue [12]. Adipose has a mesodermal origin, but white and brown adipocytes develop from separate precursor cells, through separate differentiating lineages, and by using different differentiation factors.Beige adipocytes develop from precursors of white adipocytes [13,14].Adipose cells can also originate from tumor cells-trans-differentiation of breast cancer cells into functional adipocytes was reported [15]. Although the storage of energy in lipids is evolutionarily conserved, and lipid-storing cells and proteins (FIT) are ancient [16,17], the adipose organ is evolutionarily novel to mammals [18].BAT has not been described in fishes, amphibians, reptiles, or birds, and is present only in higher mammals [19,20].The overview of the evolution of adipose tissue depots shows the gradual accumulation of features such as the way of fat storage, leptin, BAT, uncoupling protein-1 (UCP-1) in BAT, and thermoregulation in mammalian evolutionary lineage [19].Adipose organ acquired several fundamental metabolic functions since the early evolution of mammals as an adaptation to new diets and thermoregulation [18].Adipose plays a central role in the energy metabolism and maintenance of glucose homeostasis [21].Adipose associated with other organs has a diversity of additional functions and adaptations, and participate in morphogenetic processes [17]. ",
"section_name": "Mammalian adipose is a metabolic and endocrine organ evolutionarily novel to mammals",
"section_num": null
},
{
"section_content": "",
"section_name": "Similarities of mammalian adipose to tumors",
"section_num": null
},
{
"section_content": "The main similarity of adipose to tumors is its capability to almost unlimited expansion.Variations in nutrition or environmental temperature cause dramatic anatomical changes in the adipose organ.In obesity, it can increase its mass tremendously.Adipose tissue expands due to hypertrophy and hyperplasia of adipocytes.The authors stress that there are \"remarkable similarities between adipose expansion and growth of solid tumors\" [22].The adipose expansion may lead to a pathological condition, i.e. obesity and related metabolic disorders.Obesity is a risk factor for developing lipomas and other types of tumors, not only adipose tumors. ",
"section_name": "The capability of unlimited expansion",
"section_num": null
},
{
"section_content": "Adipose tumors comprise a large group of human tumors.Lipomas are the most frequent soft tissue tumors (50% of all soft-tissue masses) and are found in 2% of the population [23][24][25].The mammary gland and prostate, the other evolutionarily novel mammalian organs, are also characterized by the highest incidence of tumors [26].Lipomas are benign tumors, while liposarcomas are malignant adipose tumors with different degrees of malignancy.Liposarcomas are the most prevalent soft tissue malignancy [24,[27][28][29]. ",
"section_name": "Relatively high prevalence of mammalian adipose tumors",
"section_num": null
},
{
"section_content": "The reversible plasticity of cancer cells is well known (reviewed in [2]).Cell plasticity is defined as \"the ability of cells to change their phenotypes without genetic mutations in response to environmental cues\" [30].Neoplasms have been associated with increased plasticity, although cell plasticity was first observed during normal development. The plasticity of the adipose is remarkable and reminds that of tumors.The adipose organ can increase in size or regress, depending on the energy balance.Earlier studies suggested adipocytes transdifferentiation during cold exposure, physical exercise, lactation, and obesity [9,10].At present, the appearance of beige adipocytes in WAT depots after cold exposure or stimulation (beiging or browning of WAT) is viewed as direct transdifferentiation of white adipocytes, or as differentiation from progenitor cells.The process of WAT beiging is reversible [31][32][33]. Adipose is involved in the development of the mammary gland, the other novel mammalian organ with many tumor features.Subcutaneous adipose depots participate in mammary gland formation during lactation and pregnancy [9].Earlier data suggested that mammary adipocytes transdifferentiate into mammary epithelial cells during mammary gland development [8,9].The latest cell lineage tracing studies showed that white adipocytes in the mammary gland and skin can reversibly dedifferentiate into preadipocytes [33], and adipocyte progenitor cells can differentiate into epithelial cells of the mammary gland [34].White adipocytes can reversibly transdifferentiate into myofibroblasts and cancer-associated fibroblasts during fibrosis and cancer, and dedifferentiate in liposarcomas [33].These new data support the earlier evidence on the possibility of mammary adipocytes transdifferentiation into mammary epithelial cells [8,9].On the other hand, as already mentioned above, EMT-derived breast cancer cells can trans-differentiate into post-mitotic functional adipocytes [15].Mammary adipose controls breast cancer progression: mammary preadipocytes act locally by releasing cytokines, growth factors, and extracellular matrix components [35].Interestingly, liposarcomas are very rare in the mammary gland that is characterized by the high incidence of cancer [27,28]. ",
"section_name": "The remarkable plasticity of mammalian adipose",
"section_num": null
},
{
"section_content": "Tumor growth induces angiogenesis.Similarly, adipose expansion also induces angiogenesis.The angiogenic activity of adipocytes is connected with the secretion of pro-angiogenic molecules.The newly formed vasculature is important for adipogenesis.Angiogenesis is a rate-limiting step for adipose expansion [22,36]. ",
"section_name": "Adipose expansion induces angiogenesis",
"section_num": null
},
{
"section_content": "Like solid tumors, adipose expansion is connected with hypoxia.Hypoxia is one of the factors that cause macrophage infiltration of obese adipose tissue.The other factors are adipocyte death, chemotactic regulation, and fatty acid flux [22].Infiltrated macrophages participate in adipose tissue inflammation.Saturated fatty acids released from adipocytes are ligands for Toll-like receptor 4 complex located on macrophages.Their interaction induces inflammatory changes in macrophages, which include TNFα production.A paracrine loop involving saturated fatty acids and TNFα causes chronic inflammatory responses in adipose tissue [37].Low-grade chronic inflammation connected with obesity is a risk factor for many cancers [38]. Similar interactions between endogenous ligands and pathogen sensors occur in tumors which are also chronic inflammatory diseases.Tumor-associated macrophages are a major type of inflammatory cells infiltrating most tumors.The recruitment of immune cells and increased expression of inflammatory mediators in tumors constitute the phenomenon of tumor-elicited inflammation.Inflammation is connected with the initiation of tumors and with different stages of tumor progression [38]. ",
"section_name": "Chronic inflammation",
"section_num": null
},
{
"section_content": "Considerable changes in obese adipose tissue, including changes of extracellular matrix (ECM), adipogenesis, and metabolism, constitute adipose tissue remodeling [22,37].Tumors also undergo considerable dynamic changes and remodeling of chromatin [39], ECM [40,41], vasculature [42], and metabolism [43]. Inflammation, fibrosis, and impaired angiogenesis cause disfunction of adipose organ, which leads to obesity and related metabolic complications [44,45].The loss of function and differentiation features is also connected with tumor development (reviewed in [2]). ",
"section_name": "Remodeling and disfunction",
"section_num": null
},
{
"section_content": "Obesity-related inflammation and oxidative stress cause DNA damage in adipocytes and other tissues [46] that can lead to obesity-related carcinogenesis [47].This suggests the similarity of DNA damage mechanisms in obesity and carcinogenesis.There are even more similarities.DNA damage in obese adipocytes activates the p53 pathway [48], as it does in tumors [49].p53 negatively regulates both tumorigenesis and adipogenesis [50]. DNA damage is an initial stage of molecular processes that leads to genomic instability.Genomic instability is a feature of most tumors [51]. In hereditary cancers, mutations in DNA repair genes cause genomic instability [51].CIDE proteins involved in regulating lipid metabolism belong to the family of Cell death-Inducing DNA fragmentation Factor Alpha (DFFA)-like Effector proteins.They may participate in the DNA fragmentation step in apoptosis [52,53]. Apoptosis of adipocytes is anticipated in the stressful obese environment.However, anti-apoptotic factors such as YAP, TAZ, and Bcl2 are activated in obese adipocytes protecting them from cell death [54], a situation similar to that in tumor cells.Survivin, another potent apoptosis inhibitor, is upregulated in obesity by inflammation and oxidative stress.Survivin is also the regulator of lipid storage and metabolism.On the other hand, survivin is an oncogene expressed in most tumors.Thus, survivin is the direct molecular link between obese adipose and tumors [55]. ",
"section_name": "DNA damage and resistance to apoptosis",
"section_num": null
},
{
"section_content": "Adipose, as the central energy metabolism regulator, influences other tissues' metabolism.Adipose regulates the other tissues' metabolism according to the nutritional balance of the organism.Obesity causes systemic metabolic disorders such as insulin resistance and diabetes [7,56,57].The systemic influence of tumors on the organism has also been known since long ago [58,59]. Tumor cells have a higher rate of glucose consumption than normal cells [60][61][62].That is why tumors are called \"the glucose trap\" [61].Adipose has enhanced glucose utilization during accelerated body-fat recovery (catchup fat), which is connected with muscle-adipose glucose redistribution [63,64].The authors use the terminology \"the glucose sink\" to describe the role of enhanced de novo lipogenesis in regulating glycemia during catch-up growth [65]. Cancer can cause cachexia, a wasting syndrome.Cachexia is associated with systemic inflammation connected with tumors and tumor-induced changes in the metabolism [66,67].Brown adipose tissue and energy expenditure are increased in cachexic patients.The uncoupling protein UCP3, found in brown adipose tissue, is increased in cancer and is connected with high energy expenditure [68].Tumor-derived parathyroid hormonerelated protein (PTHrP) is involved in adipose beiging, energy-wasting, and cancer cachexia [69]. Hormone production Both adipose and tumors produce hormones.The concept of adipose as an endocrine organ is widely accepted [7,56,70,71].Adipose organ secretes several hormones (adipokines and batokines [72]) and classical cytokines, especially TNFα.The energy metabolism is regulated by adipokines leptin, adiponectin, resistin, and others [56].Leptin has an important physiological role in the central control of energy and lipid metabolism and the regulation of metabolism-immune system interplay (immunometabolism) [73].Mammalian leptin is defined as a lipostatic signal, which regulates energy balance by controlling food intake.It also regulates glucose homeostasis maintenance and participates in the regulation of immunometabolism [56,73]. The phenomenon of hormone secretion by nonendocrine tumors is known as \"ectopic\" hormone production [74][75][76].It causes unique clinical syndromes or endocrine paraneoplastic syndromes.These syndromes represent an important cause of morbidity and mortality [77].Ectopic hormones are similar to normal hormones, but in tumors, they usually are present in lower amounts per unit mass than in normal endocrine organs. miRNA production Both adipose and tumors produce miRNAs that influence other tissues.For example, adipose-derived circulating miRNAs can regulate gene expression in other tissues [78].Tumor-derived immunemodulatory miRNAs influence cancer immune surveillance and immune escape [79]. Immunosuppression Obesity and related metabolic syndrome cause negative effects on immunity [56,73,80,81].Cancer immunosuppression is also a well-known phenomenon [82][83][84][85].Obese metabolism suppresses antitumor immunity [86]. ",
"section_name": "Systemic influence in the organism",
"section_num": null
},
{
"section_content": "Ectopic lipid deposition (ELD) in skeletal muscles, heart, liver, pancreas, placenta, and kidney during obesity is a major cause of metabolism distortion [87][88][89].ELD is caused by the formation of lipid droplets in the organ's parenchymal cells; in adipocytes originated by differentiation of resident adipogenic progenitors; or in adipocytes differentiated after infiltration of organs with adipocyte progenitors from subcutaneous adipose tissue [90][91][92][93].Infiltration of adipocyte progenitors in other organs with a negative influence on these organs' functions is the most important similarity with tumor metastasis. CXCL12/CXCR4 chemokine axis participates in tumor progression and metastasis [94][95][96].Adipocyte progenitors trafficking is also regulated by the CXCL12/CXCR4 axis [93]. ",
"section_name": "Destructive infiltration in other organs and tissues",
"section_num": null
},
{
"section_content": "Thiazolidinediones (or glitazones) are used for the treatment of type 2 diabetes.By binding peroxisome proliferator-activated receptor gamma (PPARγ) they promote the maturation of adipocytes.They also suppress tumor cell invasion, migration, and invasiveness through CXCL12/ CXCR4 pathway.In addition, it was found that treatment of mice with pioglitazone (a member of the glitazone group) prevents infiltration of adipocyte progenitors in skeletal muscles [93].The other class of antidiabetic drugs-biguanides-also act as anti-carcinogens and inhibitors of tumor growth [97][98][99]. ",
"section_name": "Similar drugs may be used for the treatment of obesity and cancer",
"section_num": null
},
{
"section_content": "Obesity-related inflammation and oxidative stress cause DNA damage that can lead to obesity-related carcinogenesis [47]. Obesity and type 2 diabetes are associated with the risk of cancer and cancer-related mortality, as shown in epidemiological studies [100].The link can be related to the insulin/insulin-like growth factor (IGF) system [57,100].Throughout evolution, this system \"has integrated the control of tissue growth with metabolic status\" [57].Tumors are connected with the insulin/IGF system and systemic metabolism.The development and progression of several types of cancer are determined by the insulin/ IGF system [98].The factors that play a role in this connection include insulin resistance, hyperinsulinemia, increased levels of insulin growth factors (IGFs), hormones, and inflammatory markers [57,100,101].Using Paget's terminology of \"seed\" and \"soil\" as related to tumor metastasis [102], Holly and co-authors describe the internal milieu of obese individuals, or \"soil,\" as containing high levels of glucose, insulin, insulin-like growth factors, inflammatory cytokines, and adipokines.These authors believe that such an environment stimulates the latent lesions, the \"seeds,\" to progress to clinical cancer [57].Indeed, obese metabolism suppresses antitumor immunity [86]. The paradox of some benefits of obesity in cancer is also known: moderate overweight and early obese states can improve the survival and response to therapy [103,104]. ",
"section_name": "The connection between obesity and cancer",
"section_num": null
},
{
"section_content": "Thus, adipose, an evolutionarily young organ of mammals, shares many features with tumors.Adipose unlimited expansion is similar to tumor growth, and lipomas are the most frequent soft tissue tumors.Chronic inflammation is characteristic of obese adipose and tumors.Both adipose and tumors exert systemic metabolic and immunological influence on the organism; both participate in paracrine and endocrine interactions with other tissues; both produce miRNAs that influence other tissues; both are characterized by plasticity, induce angiogenesis and participate in morphogenetic processes.Tumors act as \"glucose trap\", and adipose during catchup fat-as \"glucose sink\".Obese adipose and tumors can cause immunosuppression; obese adipose and tumors are connected with remodeling and disfunction, with DNA damage and cell death resistance.Most important, adipose cells can metastasize into normal organs and impair their functions, similarly to malignant tumors.Finally, the same drugs and interventions are used against obesity, diabetes, ectopic lipid deposition, and tumors.Many of the common features of tumors and adipose organ are in the list of so-called \"hallmarks of cancer\" [105], and many of them are connected with the obese state of the adipose organ. As discussed earlier, other evolutionarily novel organs of mammals, such as the placenta, mammary gland, and prostate, also have many tumor features [2,5].However, evolutionarily older organs are characterized by lower cancer rates [26] and do not have (or have fewer) tumor features.The author suggested calling normal organs, which have many tumor features, the \"tumor-like organs\" [3,5].We may conclude that mammalian adipose is a tumor-like organ and obesity is a tumor-like process. ",
"section_name": "Mammalian adipose is a tumor-like organ, and obesity is a tumor-like process",
"section_num": null
},
{
"section_content": "The mammalian adipose organ's tumor features suggest its recent evolutionary origin from ancestral hereditary tumors.Following the main hypothesis, the adipose organ's origin may be represented as follows.Some diffuse mesenchymal hereditary tumors in eutherian ancestors, which produced several biologically active compounds (future adipokine hormones), acquired the capability to synthesize and accumulate fat using preexisting and evolutionarily novel genes.Accumulation of lipids inhibited the potential of progression to malignancy (\"gain fat-loose metastasis,\" [15]) and, together with future adipokine substances, was selected in ancestral Mammalia for control of energy metabolism in connection to the nutritional status of the organism, as an adaptation to new diets and thermoregulation.As a result of this evolutionary process, the evolutionarily novel mammalian organ involved in the storage and expenditure of energy with many ancestral tumor features-the adipose organ-originated. The lab of the author has already obtained the evidence in support of a hypothesis specific to the origin of mammalian adipose organ. In our previous article [4], we studied fish genes expressed in transgenic zebrafish inducible tumors, tumors after regression, and spontaneous zebrafish tumors.Among these genes, using the Orthologous Matrix (OMA) approach, we selected genes evolutionarily novel to fishes (as compared to lamprey, myxine, and other organisms in fish evolutionary lineage), and studied their human orthologs.We described many human orthologs that acquired progressive functions (such as involvement in the development of the placenta, mammary gland, lungs, neocortex, according to Gene Ontology studies), which are not encountered in fish [4].Several of those human genes with progressive functions-LEP, NOTCH1, SPRY1, PPARG, ID2, and CIDEA genes-also acquired functions connected with the adipose organ.Thus, human LEP, which encodes leptin, became the central regulator of energy metabolism in mammals.It is involved in beige/brown fat cell differentiation regulation [4,106] and lipostatic function (fish leptin is not an adipostat [107,108]).Mammalian leptin is also involved in thermoregulation [109,110]. NOTCH1 regulates adipose browning, energy metabolism, and thermogenesis [111,112]. SPRY1 is mandatory for the initiation and regulation of adipogenesis, for maintaining proliferation and differentiation of human adipose stem/progenitor cells (ASCs).It is induced in ASCs after weight loss [113,114].SPRY1 can suppress PPARG [115]. PPARG , the ortholog of the fish pparg gene, was selected in [4] because of its involvement in placenta development.Peroxisome proliferator-activated receptor gamma (PPARγ), encoded by PPARG , is the target of thiazolidinediones antidiabetic treatment, as discussed above.PPARγ participates in the differentiation of adipocytes and activation of thermogenic gene expression in brown adipocytes [116].PPARγ is a major regulator of adipocyte differentiation and function [117].It plays a role in lipodystrophy, obesity, and diabetes [118] and can downregulate LEP gene expression [104,119]. ID2 stimulates PPARγ expression, adipocyte differentiation, and adipogenesis.Its expression is elevated in adipose tissues during obesity [120]. The CIDEA gene was also found among human orthologs of novel fish genes expressed in fish tumors [4].CIDE proteins are associated with lipid droplets and regulate lipid metabolism.CIDE protein family includes CIDEA, CIDEB, and CIDEC proteins [52,53].In mice, CIDEA is a marker of brown and brite adipocytes [121].In humans, the CIDEA gene regulates adipocyte beiging [122].It means that in mammals CIDEA gene also acquired progressive functions not encountered in fish.Transcription of CIDEA gene is activated by PPARγ [52]. Each of the LEP, NOTCH1, SPRY1, PPARG , ID2, and CIDEA genes is also involved in tumor development in humans. Leptin is overexpressed in breast cancer [104] and many other types of cancer [123], has a role at different levels and participates in cancer progression.Its activation results in the activation of multiple oncogenic pathways.Leptin oncogenic functions are reinforced through crosstalk with oncogenes, e.g.NOTCH [104]. NOTCH1 has both oncogenic and tumor suppressor abilities [124,125]. SPRY1 is downregulated in some tumors and overexpressed in other tumors.Depending on the cellular context, it may serve either as a tumor suppressor or tumor promoter.SPRY1 expression is essential for induction, maintenance, and progression of tumors [126][127][128][129][130][131]. PPARγ plays oncogenic and tumor suppressor roles.PPARγ functions as a tumor suppressor in colon, lung, pancreatic, and breast cancers.A tumor-promoting role for PPARγ has been suggested in a variety of cancers as well [117]. ID family of proteins participates in the regulation of pathways essential to the progression of cancer.ID gene transcription is sensitive to signals from the cellular environment including oncoproteins.Depending on the context, ID proteins can play tumor-promoting or tumor-suppressing roles [132][133][134].The tumor-suppressive role of ID2 has been described in [135,136], and its oncogenic role-in [137]. CIDE proteins control lipid droplets' size and metabolism [53].Lipid droplets actively participate in tumor processes and accumulate in a variety of cancer cells [138,139].CIDEA controls the beiging of adipocytes [122], and beige adipocytes contribute to breast cancer progression [140].CIDEA plays an important role in human cachexia [141].CIDE proteins were originally discovered as apoptotic proteins.They induce caspaseindependent cell death in various cell types [53] that can be connected with cancer processes. Once more, orthologs of LEP, NOTCH1, SPRY1, PPARG , ID2, and CIDEA genes originated in fishes and were expressed in fish tumors.In humans, these genes acquired progressive functions not encountered in fishes, including functions connected with mammalian adipose organ, form a gene network with mutual influences, and participate in tumor processes.These genomic and transcriptomic data support the possibility of mammalian adipose origin from ancestral hereditary tumors and the tumor-like nature of mammalian adipose. The hypothesis of adipose origin by ancestral tumor neofunctionalization is also strongly supported by the experimental cytology approach: it was demonstrated that breast cancer cells can trans-differentiate, in experimental conditions of adipogenesis, into post-mitotic functional adipocytes with the loss of malignancy [15].Similarly, by trans-differentiation after the expression of evolutionarily novel genes and gene combinations involved in adipogenesis, mammalian adipocytes could originate in evolution from ancestral hereditary tumor cells. The possible origin of mammalian adipose from ancestral tumors is in correspondence with other examples of hereditary tumors, which played roles in the origin of new cell types, tissues, and organs discussed in our previous publications [1][2][3]5]. ",
"section_name": "The possible origin of mammalian adipose from ancestral mesenchymal hereditary tumors",
"section_num": null
},
{
"section_content": "The ongoing synthesis of evolutionary biology and health sciences attempts to find evolutionary roots of disease [1,142,143].If the hypothesis of the evolutionary origin of the adipose organ from the ancestral hereditary tumor is correct, it may help find new clues to obesity and cancer.Approaches developed to prevent and treat obesity may be examined to prevent and treat tumors and vice-versa.Empirically, some of such approaches are already underway [144].Cancer metabolism is currently being studied for therapeutic opportunities, along with calorie restriction interventions for the prevention and treatment of cancer.Obese adipose supports tumor growth in various ways, and interventions aimed at metabolic disorders caused by adipose expansion may also be effective against tumors. Our hypothesis may add a theoretical ground to such studies and may open new opportunities to resolve the oncological problem and the problem of the obesity epidemic.New interventions targeting LEP, NOTCH1, SPRY1, PPARG , ID2, and CIDEA gene network, in addition to what already is going on, can be designed for treatment and prevention of both obesity and tumors. ",
"section_name": "Conclusion",
"section_num": null
}
] |
[
{
"section_content": "The author is grateful to anonymous reviewers for their helpful comments. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This research work was supported by the Academic Excellence Project 5-100 proposed by Peter the Great St. Petersburg Polytechnic University, and by funding from Vavilov Institute of General Genetics, RAS. ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "Not applicable. ",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "APK is an only author.The author read and approved the final manuscript. Ethics approval and consent to participate Not applicable. Not applicable. The authors declare that they have no competing interests. • 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 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": "Abbreviations",
"section_num": null
},
{
"section_content": "",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "APK is an only author.The author read and approved the final manuscript. ",
"section_name": "Authors' contributions",
"section_num": null
},
{
"section_content": "Ethics approval and consent to participate Not applicable. ",
"section_name": "Declarations",
"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. • 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": "Competing interests",
"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
}
] |
10.21320/2500-2139-2021-14-2-225-230
|
CAR-T Cells for the Treatment of Chronic Lymphocytic Leukemia: Literature Review
|
<jats:p>Chronic lymphocytic leukemia (CLL) is the most common adult malignant lymphoid disease. Despite new highly effective targeted drugs, the prognosis of relapsed and resistant form of this disease is poor. CAR-T cell therapy using T-lymphocytes with chimeric antigen receptor (CAR) demonstrated its efficacy in the treatment of such oncohematological diseases as B-cell non-Hodgkin’s lymphomas and acute lymphoblastic leukemia. The present literature review focuses on the experience of using CAR-T cells for CLL therapy. It presents the advantages and drawbacks of this technique as well as the challenging issues to be solved for its implementation into broad clinical practice.</jats:p>
|
[
{
"section_content": "И.В.Грибкова, А.А.Завьялов КЛИНИЧЕСКАЯ ОНКОГЕМАТОЛОГИЯ идных тканях.ХЛЛ -опухоль в основном пожилых людей, 5-летняя выживаемость составляет 79,2 % [2].Прогноз оценивается преимущественно на основе цитогенетического и молекулярно-биологического исследований.Два наиболее неблагоприятных фактора связаны с плохим прогнозом: 1) изменения p53 (делеция 17p и/или мутация гена TP53) и 2) комплексный кариотип (> 3 аномалий) [1]. До недавнего времени лечение ХЛЛ первой линии заключалось главным образом в применении цитостатических препаратов, что не всегда предотвращало развитие рецидивов заболевания [3].Разработка новых препаратов, таких как ингибитор тирозинкиназы Брутона (BTK) ибрутиниб и ингибитор В-клеточной лимфомы 2 (Bcl-2) венетоклакс, привела к повышени ю эффективности терапии.Данные препараты высокоэффективны при ХЛЛ и вызывают длительные ремиссии, в т. ч. у пациентов с рецидивами после химиотерапии [4,5].Однако они дорогостоящие и требуют постоянного применения, что приводит к высокой экономической нагрузке.К тому же существуют пациенты с опухолями, резистентными к такому лечению.Только у 10-30 % пациентов, получавших венетоклакс или ибрутиниб, достигалась полная ремиссия [6,7].Кроме того, примерно у 50 % пациентов, применяющих эти препараты, рецидивы развиваются в течение 3-4 лет.Такие пациенты имеют неблагоприятный прогноз [8,9]. При ХЛЛ признается, что высоким лечебным потенциалом обладает трансплантация аллогенных гемопоэтических стволовых клеток (аллоТГСК) [10].Однако из-за преклонного возраста и множества сопутствующих заболеваний у большинства пациентов с ХЛЛ трансплантация часто становится невозможной. CAR Т-клеточная терапия, предполагающая использование Т-лимфоцитов с химерным антигенным рецептором (CAR) в настоящее время привлекает все большее внимания из-за ее многообещающих результатов.Т-клетки, несущие химерный антигенный рецептор (CAR Т-клетки), генерируются с помощью генной инженерии.На поверхности иммунокомпетентных Т-клеток пациента находится активирующий рецептор, состоящий из: 1) внецитоплазматического вариабельного фрагмента иммуноглобулина (scFv), направленного против мишени опухоли, 2) молекулы активации внутриклеточного рецептора Т-клеток (CD3ζ) и 3) молекулы положительной костимуляции (обычно CD28 и/или 4-1BB) [11].Преимущество CAR T-клеток по сравнению с обычными аутологичными T-клетками заключается в том, что они могут идентифицировать антигены независимо от главного комплекса гистосовместимости.При этом уничтожение опухолевой клетки происходит с помощью цитотоксических эффекторных механизмов. Самые эффективные CAR Т-клетки, разработанные к настоящему времени, направлены против CD19: тисагенлеклейсел и аксикабтаген силолейсел.Препараты одобрены для применения в Соединенных Штатах и Европе в 2017-2018 гг.Тисагенлеклейсел рекомендуется для лечения пациентов в возрасте до 25 лет с рефрактерным острым лимфобластным лейкозом (ОЛЛ) и взрослых пациентов с диффузной B-крупноклеточной лимфомой (ДВКЛ) при рецидивах или резистентности к стандартной иммунохимиотерапии.Аксикабтаген силолейсел зарегистрирован только для терапии ДВКЛ [11,12]. ХЛЛ был одним из первых заболеваний, при которых использовались CAR Т-клетки [13].Однако опыт применения этого метода лечения в настоящее время наиболее широкий при ОЛЛ и ДВКЛ.Доклинические и клинические данные показывают, что CAR T-клеточная терапия вполне может использоваться как способ лечения пациентов с рецидивами и рефрактерным течением ХЛЛ.Объяснением низкого уровня ответа на CAR T-терапию считается глубокая иммунная модуляция при ХЛЛ [14].Иммунные эффекторные клетки, которые необходимы для успешной иммунотерапии, такие как T-лимфоциты CD4+ и CD8+, имеют фенотипические и функциональные дефекты.Однако данные о том, что в настоящее время есть пациенты с устойчивой ремиссией в течение более 8 лет после однократной инфузии CAR Т-клеток, подтверждают, что этот подход может быть высокоэффективным по крайней мере у некоторой части пациентов с рецидивами и резистентным ХЛЛ. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Изменения Т-клеток при ХЛЛ были описаны, начиная с наблюдения, в котором отмечалось, что у пациентов с данным заболеванием увеличивается количество Т-клеток CD4+ и CD8+ [15].Далее было показано, что фенотип и функции Т-клеток заметно отличаются у пациентов с ХЛЛ по сравнению со здоровыми людьми.Так, продемонстрировано уменьшение количества наивных T-клеток и увеличение подмножеств T-клеток памяти [16].T-лимфоциты CD8+ имеют функциональное нарушение, характеризующееся неспособностью формировать иммунные синапсы с клетками-мишенями, сниженную цитотоксичность и способность к пролиферации [17].Т-клетки также отличаются повышенной экспрессией нескольких ингибирующих рецепторов на клеточной поверхности, таких как PD-1, CD160, CD244 и TIGIT [17,18].Кроме того, у пациентов с ХЛЛ увеличено количество регуляторных Т-клеток, которые ослабляют противоопухолевые иммунные ответы [19]. На измененный фенотип и функцию T-клеток CD4+ и CD8+ непосредственно влияют клетки ХЛЛ, т. к. показано, что изменения в экспрессии генов могут индуцироваться в здоровых T-клетках путем совместного их культивирования с клетками ХЛЛ [20].Также доказано, что клетки ХЛЛ индуцируют дифференцировку Т-лимфоцитов в регуляторные Т-клетки [21]. Таким образом, хотя и было показано, что Т-клетки CD4+ и CD8+ способны распознавать опухолевые клетки ХЛЛ, функциональная модуляция клоном опухоли приводит к ингибированию опосредованных Т-клетками иммунных ответов и неадекватному контролю опухоли. Эти изменения Т-клеток влияют на эффективность CAR Т-клеточной терапии и обусловливают необходимость дополнительных воздействий для повышения ответа. ",
"section_name": "ИММУНОМОДУЛЯЦИЯ Т-КЛЕТОК ПРИ ХЛЛ",
"section_num": null
},
{
"section_content": "Со времени первого сообщения об эффективности CAR Т-клеток при ХЛЛ в 2011 г. [13] были опубликованы результаты более 15 исследований применения CAR Т-клеточной терапии в общей сложности более чем у 130 пациентов с ХЛЛ [13,[22][23][24][25][26][27][28][29][30][31][32][33][34][35][36].В большинстве исследований использовались конструкции, распознающие CD19 на опухолевых клетках и содержащие домены костимуляции CD28 или 4-1BB.Пациенты, принимавшие участие в этих исследованиях, имели особенно плохой прогноз, большинство из них были с рецидивами после большого количества линий предшествующей терапии.Около половины пациентов уже получали ибрутиниб [27,28,31,[33][34][35], около 20 % -венетоклакс [31,33,35], некоторые пациенты находились в стадии рецидива после аллоТГСК [25,29].Кроме того, около 70 % больных имели изменения р53, а 60 % обследованных -комплексный кариотип.Анализ представленных данных показывает, что примерно 50-70 % пациентов с ХЛЛ имели ответ на CAR T-клеточную терапию, при этом полный ответ был получен у 20-30 % больных.Выживаемость без прогрессирования оценивается в 25 % через 18 мес.[27,37].В рандомизированном исследовании II фазы по оптимизации дозы [38,39] было показано, что более высокая доза CAR T-клеток (5 × 10 8 vs 5 × 10 7 ) дает более высокие показатели общего и полного ответов. По сравнению с данными об эффективности CAR T-клеточной терапии при ОЛЛ и ДВКЛ результаты при ХЛЛ оказываются ниже: частота полной ремиссии при ХЛЛ составляет 20-30 vs 70 % при ОЛЛ и 50 % при ДВКЛ [40,41].Однако, несмотря на то что число пациентов, у которых достигается полный ответ, невысокое, прогноз у этих больных благоприятный, с низкой вероятностью рецидивов.По этой причине в настоящее время главной задачей CAR Т-клеточной терапии у пациентов с ХЛЛ является повышение частоты полных ответов [40,42]. Поскольку предполагается, что более низкая эффективность CAR T-клеток при ХЛЛ может быть отчасти обусловлена функциональными нарушениями Т-клеток пациентов при данном заболевании, то и надежды на повышение эффективности связывают с воздействием на Т-клетки.В настоящее время для повышения эффективности используют предшествующую инфузии CAR Т-клеток химиотерапию, сопровождающуюся лимфодеплецией -устранением собственных клеток, способных снижать активность CAR T-лимфоцитов.Дело в том, что собственная иммунная система организма, и прежде всего регуляторные Т-клетки, подавляет функции Т-хелперов, цитотоксических Т-лимфоцитов и естественных киллеров.Так, в исследованиях [43,44] было показано, что лимфодеплеция приводит к созданию более благоприятной среды для пролиферации инфузированных лимфоцитов, вероятно, за счет сокращения популяции регуляторных Т-клеток, которые могут снижать активность CAR T-лимфоцитов.Это, в свою очередь, может способствовать более надежному клиническому ответу.Наиболее эффективными с точки зрения лимфодеплеции считаются схемы на основе флударабина и циклофосфамида [42]. В ряде случаев CAR Т-клеточную терапию комбинируют с ибрутинибом.Исследования in vitro [28,45] продемонстрировали способность ибрутиниба стимулировать экспансию, поддержание уровня и цитотоксичности CAR Т-клеток, а также стимулировать клеточные иммунные ответы (в частности, ибрутиниб способствует уменьшению маркеров истощения, модификации профилей секреции цитокинов, увеличению разнообразия T-клеток и др.).Комбинирование CAR T-клеток и ибрутиниба описано в исследованиях [28,33,34,46], в которых сообщается о повышении эффективности при использовании такой комбинации.В работе M.B.Geyer и соавт.[46] частота объективных ответов у пациентов, которые получали ибрутиниб до сбора Т-клеток, составляла 80 %, тогда как у всех пациентов она была только 45 %.Другое исследование [28] включало 3 пациентов, которые прекратили принимать ибрутиниб незадолго до лейкафереза, предшествующего CAR T-клеточной терапии.Ответ наблюдался у всех пациентов, включая полную ремиссию в 1 случае, несмотря на отсутствие лимфодеплеции.В 2 недавних исследованиях сообщается о результатах в двух сериях из 19 пациентов, получавших структурно разные CAR Т-клетки в сочетании с ибрутинибом.Общий уровень ответа был более 80 %, а частота полного ответа с неопределяемой минимальной остаточной болезнью превысила 90 % [33,34]. Таким образом, хотя и требуются более длительные наблюдения, представленные данные свидетельствуют о том, что добавление ибрутиниба до лейкафереза, а также его применение совместно с инфузией CAR T-клеток могут существенно улучшить эффективность CAR Т-клеточной терапии. ",
"section_name": "CAR T-лимфоциты в лечении ХЛЛ ДАННЫЕ ОБ ЭФФЕКТИВНОСТИ CAR Т-КЛЕТОЧНОЙ ТЕРАПИИ ПРИ ХЛЛ",
"section_num": null
},
{
"section_content": "В исследованиях [47,48] сообщается, что наивные T-клетки (TN) и центральные клетки памяти (TCM) превосходят по противоопухолевой активности эффекторные клетки памяти (TEM) и эффекторные Т-клетки (TE).Они показывают бóльшую способность к пролиферации и цитотоксичности.К сожалению, CAR T-клеточные продукты пациентов с ХЛЛ всегда демонстрируют низкое соотношение TN/TE и, таким образом, не могут быть достаточно эффективными.Интерлейкины-7/15 (IL-7/IL-15) могут индуцировать дифференцировку T-клеток в TN и TCM.Следовательно, можно использовать IL-7/IL-15 в качестве стимуляторов в процессе производства CAR T-клеток [48]. Кроме того, наблюдается некоторая кооперация при одновременном использовании TN и TCM, а определенное соотношение Т-клеток CD4+/CD8+, например 1:1, приводит к лучшим результатам терапии [49].К сожалению, соотношение Т-клеток CD4+/CD8+ у пациентов с ХЛЛ, получавших предшествующую терапию, далеко от соотношения 1:1, а популяции И.В.Грибкова, А.А.Завьялов ",
"section_name": "ДОПОЛНИТЕЛЬНЫЕ ВОЗМОЖНОСТИ ПОВЫШЕНИЯ ЭФФЕКТИВНОСТИ CAR T-КЛЕТОЧНОЙ ТЕРАПИИ ПРИ ХЛЛ",
"section_num": null
},
{
"section_content": "Т-клеток содержат в основном TEM и ТЕ.Следовательно, для повышения эффективности CAR T-клеточной терапии при ХЛЛ желательны перераспределение соотношения Т-клеток CD4+/CD8+ и выбор TN и TCM путем сортировки Т-клеток в соответствии с различным фенотипом. Опубликованы данные исследований CAR Т-клеточной терапии, нацеленной на опухолевые мишени, отличные от CD19: CD23, ROR1, κ-и λ-цепи CD20, Fc-рецептор иммуноглобулина M (FcμR) и др.[41].Показано, что некоторые из этих мишеней могут оказаться более перспективными для терапии ХЛЛ [41]. ",
"section_name": "КЛИНИЧЕСКАЯ ОНКОГЕМАТОЛОГИЯ",
"section_num": null
},
{
"section_content": "Синдром цитокинового шторма (СЦШ), В-клеточная аплазия, нейротоксичность и инфекции являются частыми побочными эффектами CAR T-клеточной терапии при ХЛЛ [50][51][52].Частота СЦШ относительно высокая при ХЛЛ и составляет почти 83 %, наиболее часто СЦШ встречается у пациентов, которые ранее принимали ибрутиниб [31].Однако комбинация ибрутиниба и CAR Т-клеток, по-видимому, связана с более низкой частотой возникновения СЦШ ≥ III степени тяжести [33,34].При цитокиновом шторме возможно поражение с нарушением функции разных органов и систем.Клинические симптомы при СЦШ могут быть различными: лихорадка, нарушение дыхательной функции, тахикардия, гипотензия, генерализованные отеки, нарушение сознания, генерализованная лимфаденопатия, гепатоспленомегалия, эритематозная или зудящая сыпь.Тяжесть течения СЦШ варьирует и может достигать уровня полиорганной недостаточности с летальным исходом [26].Для лечения применяются антагонист рецептора IL-6 тоцилизумаб и кортикостероиды [53]. Поскольку CD19 широко экспрессируется на B-клетках, анти-CD19 CAR T-клетки могут уничтожать как клетки ХЛЛ, так и нормальные B-лимфоциты, вызывая аплазию B-клеток, которая характеризуется дефицитом B-клеток и иммуноглобулина (эффект «on target/off tumor») [24].Возможным решением этой проблемы является заместительная терапия иммуноглобулином для внутривенного введения [54]. У некоторых пациентов с ХЛЛ отмечается неврологическая токсичность, механизм развития которой остается неясным.Нейротоксичность I-III и IV-V степени тяжести при ХЛЛ встречается у 29 и 4 % пациентов соответственно [52].Как правило, ее симптомы включают делирий, афазию, судороги и т. д.Следует отметить, что большая часть неврологической токсичности обратима и может спонтанно разрешаться без какого-либо лечения.При тяжелой нейротоксичности рекомендуется использовать высокие дозы кортикостероидов.Кроме того, возможно применение анти-IL-6-антител (тоцилизумаб) при неврологической токсичности с одновременным СЦШ [55]. Инфекции также бывают тяжелыми побочными эффектами при CAR T-клеточной терапии из-за гипогаммаглобулинемии и нейтропении.Частота возникновения инфекционных осложнений III сте-пени тяжести и выше при ХЛЛ составляет 21-25 %.Бактерии -наиболее частая причина, но также встречаются вирусные и грибковые инфекции [50].Факторами высокого риска инфекции являются число (> 4) предыдущих схем противоопухолевого лечения, высокая доза CAR T-клеток и высокая степень тяжести СЦШ/нейротоксичности [50].При возникновении инфекционных осложнений применяют антибиотики, противогрибковые и противовирусные средства. ",
"section_name": "ДАННЫЕ О БЕЗОПАСНОСТИ CAR T-КЛЕТОЧНОЙ ТЕРАПИИ ПРИ ХЛЛ",
"section_num": null
},
{
"section_content": "Несмотря на то что терапия на основе CAR Т-клеток может быть очень эффективной, она имеет существенные недостатки.Во-первых, получение аутологичного продукта может быть затруднено из-за недостаточного количества Т-клеток, особенно у пациентов с большим объемом предшествующего противоопухолевого лечения.Во-вторых, достаточно длительное время производства CAR T-клеток делает лечение невозможным у пациентов с агрессивными злокачественными опухолями.Кроме того, CAR Т-клеточная терапия связана с серьезными побочными эффектами.Одной из основных проблем также является высокая стоимость производства аутологичных Т-клеток индивидуально для каждого пациента.Общие затраты могли бы быть снижены за счет использования аллогенных Т-клеток.Однако подобная терапия может обусловить риск опасных для жизни осложнений, связанных с реакцией «трансплантат против хозяина». ",
"section_name": "ПРОБЛЕМЫ, СВЯЗАННЫЕ С CAR Т-КЛЕТОЧНОЙ ТЕРАПИЕЙ",
"section_num": null
},
{
"section_content": "CAR Т-клеточная терапия является перспективным и активно развивающимся направлением в онкологии и гематологии, особенно при злокачественных опухолях с неблагоприятным прогнозом.Опыт применения данной терапии при ХЛЛ в настоящее время отстает от такового при ОЛЛ или ДВКЛ, поскольку доклинические и клинические результаты демонстрируют более низкую эффективность CAR Т-терапии при ХЛЛ.Данный факт связывают с наличием у больных ХЛЛ иммунной модуляции, снижающей эффективность CAR Т-клеточной терапии.Однако последние научные разработки в этой области показали, что существуют подходы, позволяющие эффективно применять аутологичные CAR Т-клетки и у пациентов с ХЛЛ.Так, например, комбинация CAR T-клеток с ибрутинибом, проведение предшествующей инфузии CAR Т-клеток лимфодеплеции, а также дополнительное использование IL-7/IL-15 в качестве стимуляторов в процессе производства CAR T-клеток представляются многообещающими вариантами успешной терапии ХЛЛ. Несмотря на то что многие проблемы до сих пор не решены, быстрое продвижение в понимании и развитии технологий клеточной иммунотерапии приведет к появлению новых терапевтических способов, которые, возможно, окажутся прорывными в лечении пациентов с ХЛЛ. ",
"section_name": "ЗАКЛЮЧЕНИЕ",
"section_num": null
}
] |
[
{
"section_content": "Авторы заявляют об отсутствии конфликтов интересов. Исследование не имело спонсорской поддержки. ",
"section_name": "CAR T-лимфоциты в лечении ХЛЛ",
"section_num": null
},
{
"section_content": "",
"section_name": "CAR T-лимфоциты в лечении ХЛЛ",
"section_num": null
},
{
"section_content": "Авторы заявляют об отсутствии конфликтов интересов. ",
"section_name": "КОНФЛИКТЫ ИНТЕРЕСОВ",
"section_num": null
},
{
"section_content": "Исследование не имело спонсорской поддержки. ",
"section_name": "ИСТОЧНИКИ ФИНАНСИРОВАНИЯ",
"section_num": null
},
{
"section_content": "",
"section_name": "ВКЛАД АВТОРОВ",
"section_num": null
},
{
"section_content": "",
"section_name": "ЛИТЕРАТУРА/REFERENCES",
"section_num": null
}
] |
10.1371/journal.pone.0167572
|
Yessotoxin, a Marine Toxin, Exhibits Anti-Allergic and Anti-Tumoural Activities Inhibiting Melanoma Tumour Growth in a Preclinical Model
|
Yessotoxins (YTXs) are a group of marine toxins produced by the dinoflagellates Protoceratium reticulatum, Lingulodinium polyedrum and Gonyaulax spinifera. They may have medical interest due to their potential role as anti-allergic but also anti-cancer compounds. However, their biological activities remain poorly characterized. Here, we show that the small molecular compound YTX causes a slight but significant reduction of the ability of mast cells to degranulate. Strikingly, further examination revealed that YTX had a marked and selective cytotoxicity for the RBL-2H3 mast cell line inducing apoptosis, while primary bone marrow derived mast cells were highly resistant. In addition, YTX exhibited strong cytotoxicity against the human B-chronic lymphocytic leukaemia cell line MEC1 and the murine melanoma cell line B16F10. To analyse the potential role of YTX as an anti-cancer drug in vivo we used the well-established B16F10 melanoma preclinical mouse model. Our results demonstrate that a few local application of YTX around established tumours dramatically diminished tumour growth in the absence of any significant toxicity as determined by the absence of weight loss and haematological alterations. Our data support that YTX may have a minor role as an anti-allergic drug, but reveals an important potential for its use as an anti-cancer drug.
|
[
{
"section_content": "Yessotoxins (YTXs) are a group of lipophilic marine exotoxins produced by the dinoflagellates Protoceratium reticulatum, Lingulodinium polyedrum and Gonyaulax spinifera [1][2][3].The group of YTXs is composed of close to 90 known analogs (YTX, homoYTX, hydroxyYTX, carboxyYTX and desulfoYTX among others) [4], however, the chemical structures for most of these compounds remain unclear [1].Although no lethality was observed after oral administration in mice (doses up to 54mg/Kg), YTXs were reported to be lethal after intraperitoneal injection causing restlessness, dyspnoea, shivering, jumping and cramps albeit at relatively high levels of median lethal dose (LD 50 ) values ranging from 80 to 750μg/Kg depending on the mouse strain used [5].YTX is the most toxic among all the analogous, nevertheless, the toxicity following intraperitoneal injection or oral administration at sublethal doses does not induce neither macroscopic nor histological alterations, even in the cardiac muscle [6,7].Another study, however, reports some moderate changes and damage in the myocardium that are reversible in the long-term [8,9]. The role of YTX compounds as marine seafood contaminant toxins is presently debated.Based on the toxicity observed in mouse bioassays and their coexistence with other marine toxins (okadaic acid and dinophysistoxins) they have been included in the list of marine toxins [8].While the European Food Safety Authority (EFSA) sets an Acute Reference Dose (ARfD) of 25μg YTX equivalents/Kg body weight, the European Union has established a limit of 3.75mg YTX/Kg of shellfish meat, as a prevention measure [10].Yet, it remains a fact that the biological activity of YTXs in the environment is incompletely understood. In order to better understand the mechanism of action of YTXs some of their activities have been studied in vitro in previous works.An intriguing finding revealed a cytotoxic effect of YTX described initially in rat glioma cells [11] and hepatocytes [12].Subsequent studies performed in BE(2)-M17 neuroblastoma demonstrated that YTX induces apoptotic cell death [13].Likewise, tumoural K-562 lymphocytes were shown to be YTX-sensitive dying by apoptosis, while normal human lymphocytes proved to be YTX-resistant [14,15].In some mouse cell lines autophagy activity was found after YTX exposure [16].However, no in vivo studies testing the cytotoxic effects of YTX in the treatment of tumours have been reported so far. In addition to cytotoxicity, anti-asthmatic and anti-allergic effects have been proposed for the YTX group, albeit these therapeutic effects are not completely understood [17,18].The interaction of allergens and allergen-specific IgE with the high-affinity IgE receptor (FcεRI) represents the key event in type I hypersensivity allergic reactions triggering a signalling cascade enabling rapid release of multiple inflammatory mediators such as histamine from mast cells (MCs) and basophils [19].MCs are derived from CD34 + /c-kit + progenitor cells and play a crucial role in the pathogenesis of allergy, inflammation and many chronic inflammatory processes [20].However, no studies examining directly the effect of YTX on the viability and cellular activation as well as the degranulation of MCs have been reported so far.We therefore sought to examine the activities of YTX on MC biology.Our results show that the anti-allergic effect is minor, while the toxin profoundly inhibits the growth of tumour MCs and some other tumour cells, which undergo apoptosis, while primary bone marrow derived MCs were YTX resistant.We also studied the cytotoxic properties of YTX in vivo using the well-established B16F10 melanoma model in mice to clarify the capacity of this molecule as an anti-cancer compound. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "YTX (purity !99%) dissolved in methanol was obtained from Cifga Laboratorio (Lugo, Spain).4-Nitrophenyl N-acetyl-β-D-glucosaminide (pNAG), propidium iodide solution, MgCl 2 , glucose, DNP-human serum albumin (DNP-HSA) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were from Sigma-Aldrich (Germany).Annexin V-FITC and 10X AnnexinV Binding Buffer were from BD Pharmingen (France).HEPES was from Gibco (Life Technologies).NaCl was from VWR (Belgium).KCl and Triton X-100 were from Euromedex (France).CaCl 2 was from Prolano (Paris, France).Bovine serum albumin (BSA) was from PAA (Austria).Mouse monoclonal anti-DNP IgE has been described previously [21][22][23]. ",
"section_name": "Reagents",
"section_num": null
},
{
"section_content": "The C57/BL6 strain was raised and maintained at the animal facilities of the Center of Research on Inflammation.All experiments were performed in accordance with the national ethical guidelines and with the approval of local authorities of the Comite ´d'E ´thique Expe ´rimentation Animale Bichat-Debre ´.Animals were in cages with the following dimensions: 30x20x13 (LxWxH) and separated by three groups (Untreated, Vehicle and Yessotoxin group).Each cage was labeled with a card indicating the study, group and animal number.A maximum of 3 animals of the same group were housed in the same cage.Mice were maintained in a specific pathogen-free facility with a 12-h light/12-h dark cycle.Sterile food and sterilized pure water was provided ad libitum during the whole procedure. ",
"section_name": "Animals",
"section_num": null
},
{
"section_content": "Bone marrow-derived mast cells (BMMCs) were derived from the bone marrow of C57BL/6 mice.The mice were sacrificed by CO 2 inhalation and bone marrow cells were cultured in IMDM containing 15% fetal bovine serum (FBS) (Gibco, Life Technologies), 1mM sodium pyruvate (GE Healthcare), 100μM non-essential amino acids (Gibco, Life Technologies), 100UI/mL penicillin, 100μg/mL streptomycin (Gibco, Life Technologies) and 0.05mM 2-mercaptoethanol !99% (Gibco, Life Technologies).The medium is supplemented with 10ng/mL interleukin-3 and 10ng/mL Stem Cell Factor (both from Miltenylbiotec).Cells were cultured in an atmosphere containing 5% CO 2 .After five weeks in culture more than 95% of the cells were MCs assessed by toluidine blue staining. RBL-2H3 and B16F10 melanoma (kindly provided by Dr. Thiago Maciel, Imagine Institute, Paris, France) cell lines were grown at 37˚C in DMEM-Glutamax High Glucose medium (Gibco, Life Technologies) containing 10% FBS, 100UI/mL penicillin, 100μg/mL streptomycin (Gibco, Life Technologies) and for B16F10 additionally 1mM sodium pyruvate (Gibco, Life Technologies), 100μM non-essential amino acids and 0.05mM 2-mercaptoethanol !99% (Gibco, Life Technologies) in an atmosphere containing 5% CO 2 .The MEC1 cell line (kindly provided by Dr. Pierre Launay, INSERM U1149, Paris, France) was maintained at 37˚C in RPMI-Glutamax medium (Gibco, Life Technologies) supplemented with 10% FBS, 100UI/mL penicillin and 100μg/mL streptomycin (Gibco, Life Technologies) in an atmosphere containing 5% CO 2 . ",
"section_name": "Cells and cell lines",
"section_num": null
},
{
"section_content": "Cells (2x10 6 /mL) were incubated with monoclonal anti-DNP IgE ascites fluid (1:10,000 final dilution) overnight.After washing, cells were resuspended in 500μL Tyrode's buffer (20mM HEPES pH 7.2, 137mM NaCl, 5mM KCl, 1mM MgCl 2 , 1,8mM CaCl 2 , 5,6mM glucose, 0,5mg/ mL BSA).Cells were then treated with indicated concentrations of YTX or vehicle (methanol) for 30 min and 1h before stimulation for 45min with DNP-HSA in the presence or absence of YTX as indicated.The reaction is stopped by immersing cells on ice for 10min.The percent released β-hexosaminidase into the supernatant was then determined as previously described [24], after subtracting background release of non-stimulated cells.100% values were determined after addition of 0.5% Triton X-100 to lyse the cells. ",
"section_name": "β-hexosaminidase release measurement",
"section_num": null
},
{
"section_content": "Cell viability was measured using the MTT test.This assay measures mitochondrial function by determining the quantity of formazan formed after conversion of the soluble MTT dye by active mitochondrial dehydrogenases in live cells.Briefly, after incubation with YTX cells (0.5x10 6 /mL) were washed in 500μL Tyrode's buffer before addition to MTT (250μg/ mL) and incubation for 30min at 37˚C [14,25,26].After incubation cells were centrifuged and resuspended in 100μL H 2 O and sonicated.Final optical density obtained from formazan formation was measured at 595nm with a multi-mode plate reader (Infinite M200, Tecan 1 ).Cells lysed in 10% Triton X-100 were used as 100% positive control for death cells. Apoptosis and necrotic cell death were determined by flow cytometry using AnnexinV-FITC/propidium iodide (PI) staining.Cells (1x10 6 cells/mL) were incubated with different concentrations of YTX for indicated times.Cells were washed with Tyrode's buffer, resuspended in 100μL 1X AnnexinV Binding Buffer and stained by adding 5μL of AnnexinV-FITC (BD Pharmingen) for 15min at RT as well as of 2.5μg/mL PI solution (Sigma-Aldrich).Flow cytometry data were acquired on a Fortessa flow cytometer (BD Biosciences) and analysed using FlowJo software (TreeStar).Live cells were considered both Annexin V-FITC and PI negative; early apoptotic cells were considered Annexin V-FITC positive and PI negative, late apoptotic cells were considered both Annexin V-FITC and PI positive and necrotic cells were considered Annexin V-FITC negative and PI positive. ",
"section_name": "Determination of cellular viability and apoptosis/necrosis",
"section_num": null
},
{
"section_content": "Anti-cancer effect of YTX in vivo was studied using the B16F10 melanoma mouse model.A total of 2x10 5 B16F10 melanoma cells in 100μL PBS were injected subcutaneously into the flank of C57BL/6 females (7 weeks) inducing within 5 to 10 days a palpable tumour of about 50mm 3 [27].At this time point, mice were treated with 100μg/Kg of YTX or vehicle administrated subcutaneously in close vicinity to the site of tumour development.Additional lower doses of YTX (20μg/Kg and 10μg/Kg) were administrated subcutaneously as indicated.As the B16 melanoma is a fast growing tumour, its volume was determined daily until sacrifice by the following formula for solid tumours: volume = (length x width 2 )/2 [28,29].Treatment time was kept to a minimum of 5 days and mice were euthanized by CO 2 inhalation (5 L/min) for 10 minutes at day 12 allowing a maximum of 7 days of tumour growth before they reached necrotic or ulcerating stages as previously recommended [27].Signs like no interest on cage exploration, weight and/or appetite loss, difficulty in breathing and loss of coordination were evaluated two times per day.Additional signs of toxicity such us difficulty to eat, drink, walk or groom, distress or moribund signs or any other signs of systemic toxicity were also monitored for immediate euthanization.None of the animals suffered from any of these signs during the procedure, therefore, no animals were euthanized during the study.The maximal tumour volume achieved was in the range or lower of previously published data [30][31][32][33][34].The weight loss at the day of sacrifice considering tumour density [35] was less than 20%.A blood sample was collected before sacrifice and immediately analysed in the Melet Schloesing Haematology Analyser for haematological parameters determination including lymphocytes, monocytes, neutrophils/granulocytes, erythrocytes and platelets.All experiments were performed in accordance with the national ethical guidelines and with the approval of local authorities of the Comite ´d'E ´thique Expe ´rimentation Animale Bichat-Debre ´. ",
"section_name": "B16F10 mouse model of subcutaneous melanoma",
"section_num": null
},
{
"section_content": "At day of sacrifice 100μL of RediJect 2-DG Fluorescent Imaging Probe (Perkin Elmer 1 ) were injected intravenously to visualize glucose uptake known to be directly related to cell proliferation and hence tumour growth.After 3h, animals were anesthesized with 5mg Ketamine (Ketamine 50mg, Virbac 1 ) and 20mg Xilacin (Rompun 2%, Bayern 1 ) diluted in PBS and injected intraperitoneally.Luminiscence images were captured using a FX Pro (Kodak) and analyzed with Carestream MI software. ",
"section_name": "Glucose uptake measurement in B16F10 mouse model of subcutaneous melanoma",
"section_num": null
},
{
"section_content": "One-way ANOVA was employed for comparison of significant differences among groups.A probability level of 0.05 or smaller was used for statistical significance. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "To analyse whether the reported anti-allergic and anti-asthmatic activity of YTX involved its ability to inhibit MC degranulation we evaluated the effect of YTX on the release of granulestored β-hexosaminidase in the RBL-2H3 MC line and in primary BMMCs.IgE-sensitized RBL-2H3 cells and BMMCs were treated with 10 or 30 nM YTX for 30 or 60min before stimulating them with specific antigen (DNP-HSA) for 45min in the presence or absence (removal) of YTX.In RBL-2H3 cells no significant effect was observed at low and high antigen doses (10ng/mL and 1000ng/mL) while at more optimal stimulatory doses (100ng/mL), a slight decrease in the degranulation response was observed at 30nM of YTX, particularly when the toxin remained present during the stimulation period (Fig 1A).A decrease of the degranulation response was also observed in BMMCs after 1h incubation of YTX, independently of whether it had been removed before stimulation or not (Fig 1B, right panel).The inhibitory effect required prolonged preincubation with YTX as it was only observed after pretreatment for 1h, while no effect was observed after 30min (Fig 1B, left panel).Although these results demonstrate that YTX decreases β-hexosaminidase release, its effect was not very potent reaching with a maximum inhibition of 25% in both BMMCs and RBL cells.It also appeared to be dependent on the dose of antigen in RBL-2H3 cells disappearing at non optimal stimulatory doses (Fig 1A). As YTX was shown previously to be toxic to tumour cells at relatively low concentrations, we performed control experiments to evaluate whether the toxin had any effect on MC viability.Fig 2 shows that using the MTT assay YTX did not induce any cytotoxic effect neither in BMMCs nor in RBL-2H3 after short (30min) incubation times.However, after 1h of incubation time a slight but significant decrease or tendency to decrease in cell viability was observed in RBL-2H3 cells at the high YTX concentrations (30, 50 and 100nM).This effect became highly prominent after 24, 48 and 72h suggesting that the toxin exhibited significant toxicity towards the RBL-2H3 MC cells line.Surprisingly, the effect was different when BMMCs were examined as no significant toxicity of YTX could be observed.Only with the highest concentration of YTX (100nM) a tendency for some toxicity was observed at 48h and 72h of treatment.Taken together, these results indicate that YTX has a clear cytotoxic effect for the MC tumour line, while primary MCs were highly resistant to YTX induced toxicity. The pathway by which YTX induced cell death in RBL-2H3 cells remained unclear.We therefore used an Annexin V-FITC/PI staining assay to determine whether YTX-induced apoptosis or necrosis in these cells.The assay allowed to distinguish viable (Annexin V-FITC -/PI -), early apoptotic (Annexin V-FITC +/ PI -) and late apoptotic or necrotic cells (Annexin V-FITC +/PI +).Fig 3 shows, that upon treatment of RBL-2H3 cells with different doses of YTX the cells lost cell viability in a dose dependent manner undergoing a transition from early apoptosis after 24h to late apoptotic cells, which become highly prominent at the later time points of treatment.Some necrotic cells appeared at early time points and they significantly increased before 72h.The apoptosis induction is clearly dose dependent becoming rapidly apparent at 30nM, while the lower dose of 10nM only started to show some apoptotic effects at 72h, albeit this was not significant.Taken together these results indicate that YTX induces cell death by apoptosis. The observed results corroborate previous works confirming the anti-tumour activity of YTX [14,15].In order to study the most suitable cell line for the in vivo experiment we selected two different cell lines, MEC1 and B16F10, both of which have been used as a tumour in vivo mouse model in our laboratory.MEC1 is a human cell line obtained from a patient with Bchronic lymphocytic leukemia (B-CLL) in prolymphocytoid transformation [36], whereas B16F10 is a mouse melanoma cell line [27].The cytotoxic effect of YTX treatment was studied in both cell lines using the same kinetics as above for RBL-2H3 cells.Fig 4A shows, that YTX induces a significant decrease in cell viability at 24h of treatment at 10, 30 and 100nM doses, in MEC1 cells.The cytotoxic effect of YTX was even stronger after 48h causing a significant decrease in cell viability (around 50%) at all YTX concentrations but cytotoxicity did not decrease further at 72h of incubation with YTX.For B16F10 cells the results show that YTX caused a progressive decrease in cell viability with essentially no remaining viable cells after 72h of incubation with YTX (Fig 4B).This effect was observed at all treatment doses studied.Even the smallest dose decreased viability by up to 90%.These results were confirmed when cells were stained with Annexin V-FITC and PI (Fig 5).Results showed that already after 24h a significant proportion exhibited early signs of apoptosis and that this proportion increased significantly at later time points. The above results indicate that YTX induces a stronger cytotoxicity towards B16F10 cells as compared to the MEC1 cell line.We therefore selected the B16F10 mouse tumour model to test the YTX anti-tumour effect in vivo.To this end B16F10 cells were injected subcutaneously into the flanks of the mice and allowed to grow until appearance of a small palpable tumour bleb with a size of about 50 mm 3 .Tumour treatment was started with an initial injection at high dose (100μg/Kg) in immediate tumour vicinity followed by 4 injections at lower doses at days 2, 3, 4 and 5. ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "The interest in medical applications for algal toxins and in particular of YTX due to its low oral toxicity has grown in recent years.One of the first potential roles discovered was the antiallergic role of this compound [37].This could relate to the capacity of YTX to activate cellular phosphodiesterases (PDEs known to play a role in the activation of basophils and MCs) [17,38,39].As no systematic evaluation on the capacity of YTX on MC degranulation has been reported we studied the YTX effect on MCs degranulation using primary BMMCs and the RBL-2H3 cell line.Our results demonstrate that although YTX causes a significant decrease of β-hexosaminidase release, this effect appears minor reaching a maximum of 25% inhibition in BMMCs and RBL-2H3 cells being observed in the RBL-2H3 MC line only at optimal antigen doses. Furthermore, the present paper also demonstrated that YTX is a secondary metabolite showing an important cytotoxic effect towards the RBL-2H3 cell line at low doses in the 30 to 100nM range.Interestingly non-tumoural MCs show an extraordinary resistance as no important cytotoxic effects were observed at doses of up to 50 nM with some cytotoxicity becoming apparent at 100nM, but only after long-term incubation (72h).In addition to RBL-2H3 cells YTX exhibited also a high cytotoxic activity against MEC1 and B16F10 cell lines confirming several other studies including a National Cancer Institute human tumour cell line screen, which showed high toxicity to many tumour cell lines in the nanomolar range [18].In particular, melanoma cell lines were the most sensitive to YTX, followed by lung, colon, leukemia, mammary, ovarian, central nervous system and renal cell lines [4,18].On the other hand, low toxicity was generally observed in primary cells.Thus, previous studies in fresh human lymphocytes and K-562 lymphoma cells showed that they were, respectively, resistant and sensitive to YTX [14,15].Differential effects of YTX also showed that it does not induce cell death features in a lymphoblastoid cell line, whereas it induced apoptosis in the lymphocytic cell line K-562 [40]. Three different cell death mechanisms can be induced by YTX.The apoptotic pathway including caspase 3 activation was first described in the BE(2)-M17 neuroblastoma cell line [13], whereas paraptosis (BC3H1 myoblast cells) [41] and autophagy (human glioma and K-562 cells) were more recently described [15,41,42].Previous work also described YTX apoptosis induction in K-562 cell line (other by activating caspase 3 and 8 (extrinsic apoptotic pathway hallmark) and decreasing anti-apoptotic Bcl-2 protein levels [15].In our present manuscript the apoptotic cell death pathway was found to be the major cause of death in RBL-2H3 or B16F10 cell lines.Indeed, YTX rapidly induces exposure of phosphatidylserine as one of the early signs of apoptosis ending in a late apoptotic stage characterized by the uptake of PI in RBL-2H3 cell line.These data suggest, while YTX can induce multiple cell death pathways, including apoptosis and the recently described paraptosis and autophagy, the apoptosis pathway seems to be the primary pathway in RBL2H3 and B16F10 cells. Among three cell lines tested in the present study, B16F10 was shown to exhibit the highest sensitivity to YTX, followed by RBL-2H3 and MEC1.The B16F10 is a very aggressive murine melanoma cell line [43] and its high YTX sensitivity is in accordance with the results obtained in a previous wide scale screening, which showed high sensitivity of human melanoma cell lines to the compound [4].Malignant melanoma is the sixth most common cancer in the US [27] calling for novel drugs/therapies to effectively treat melanoma patients.Cutaneous melanomas are generated from skin melanocytes and albeit recent progress has been made through the use of checkpoint inhibitors, the effectiveness of the treatment is highly variable and depends on the patient [27,[44][45][46].Based on the significant in vitro efficacy of YTX in inducing cell death of B16F10 melanoma cells and the fact that YTX has never been evaluated as an anti-cancer drug in vivo we examined its efficacy in vivo in the B16F10 mouse melanoma model.Our results showed that local subcutaneous administration of YTX in the close vicinity to established tumour bleb resulted in a marked reduction in melanoma growth.One high dose bolus injection of 100μg/Kg followed by 4 additional low dose injections (20 and 10μg/ Kg) were sufficient to reduce by 82% the B16F10 tumour melanoma volume after 12 days without affecting animal weight and haematological parameters.These results are encouraging and can be compared with other local therapeutic strategies tested in B16F10 melanoma murine model.Firstly, YTX in vitro cytotoxicity in B16F10 cells is 1000-fold more potent than the observed with the H-15 cyclic pentapeptide [47].Another comparison includes the COOH-Terminal Peptide of Platelet Factor-4 Variant (CXCL4L1/PF-4var 47-70 ), which when injected intra-tumourally was also described as a successful therapy in B16F10 melanoma growth in vivo [48].Tumour growth inhibition due to CXCL4L1/PF-4var 47-70 after 16 days treatment was about 60% but treatment started already at the time of inoculation with tumour cells.YTX treatment seems equivalent to therapeutic effects achieved with subcutaneous vaccination of a viral cocktail expressing a C-terminal peptide of human telomerase transcriptase, an enzyme expressed in more than 85% of human tumour cells but rarely in normal cells [33].Therefore, the present work demonstrates, for first time, the activity of YTX as an anticancer compound in vivo making it a compound with a great interest in cancer therapy. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "Dr. Araceli Tobio Ageitos was supported by a postdoctoral fellowship from Fundacio ´n Juana de Vega, Spain.Dr. Iris Madera-Salcedo was supported by an International collaboration grant between ANR France (ANR-12-ISV3-0006-01) and Conacyt Mexico (Conacyt-ANR 188565).This research project has been supported by the Investissements d'Avenir programme ANR-11-IDEX-0005-02, Sorbonne Paris Cite, Laboratoire d'excellence INFLAMEX, and DHU Fire.This work was also supported by the COST Action BM1007 (Mast cells and basophils-targets for innovative therapies) of the European Community. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "All relevant data are within the paper. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Conceptualization: AT AA LMB UB. ",
"section_name": "Author Contributions",
"section_num": null
},
{
"section_content": "",
"section_name": "Data curation: AT.",
"section_num": null
}
] |
10.1371/journal.pone.0096238
|
Hyper-Activation of Notch3 Amplifies the Proliferative Potential of Rhabdomyosarcoma Cells
|
Rhabdomyosarcoma (RMS) is a pediatric myogenic-derived soft tissue sarcoma that includes two major histopathological subtypes: embryonal and alveolar. The majority of alveolar RMS expresses PAX3-FOXO1 fusion oncoprotein, associated with the worst prognosis. RMS cells show myogenic markers expression but are unable to terminally differentiate. The Notch signaling pathway is a master player during myogenesis, with Notch1 activation sustaining myoblast expansion and Notch3 activation inhibiting myoblast fusion and differentiation. Accordingly, Notch1 signaling is up-regulated and activated in embryonal RMS samples and supports the proliferation of tumor cells. However, it is unable to control their differentiation properties. We previously reported that Notch3 is activated in RMS cell lines, of both alveolar and embryonal subtype, and acts by inhibiting differentiation. Moreover, Notch3 depletion reduces PAX3-FOXO1 alveolar RMS tumor growth in vivo. However, whether Notch3 activation also sustains the proliferation of RMS cells remained unclear. To address this question, we forced the expression of the activated form of Notch3, Notch3IC, in the RH30 and RH41 PAX3-FOXO1-positive alveolar and in the RD embryonal RMS cell lines and studied the proliferation of these cells. We show that, in all three cell lines tested, Notch3IC over-expression stimulates in vitro cell proliferation and prevents the effects of pharmacological Notch inhibition. Furthermore, Notch3IC further increases RH30 cell growth in vivo. Interestingly, knockdown of Notch canonical ligands JAG1 or DLL1 in RMS cell lines decreases Notch3 activity and reduces cell proliferation. Finally, the expression of Notch3IC and its target gene HES1 correlates with that of the proliferative marker Ki67 in a small cohort of primary PAX-FOXO1 alveolar RMS samples. These results strongly suggest that high levels of Notch3 activation increase the proliferative potential of RMS cells.
|
[
{
"section_content": "Pediatric rhabdomyosarcoma (RMS) is a skeletal musclederived soft-tissue sarcoma affecting children and adolescents.It accounts for approximately 50% of all pediatric soft-tissue sarcomas and for 7-8% of all childhood malignancies [1].Pediatric RMS includes two major histological subtypes, embryonal and alveolar [2].Embryonal RMS has a favorable prognosis with survival rates of about 90% when nonmetastatic.Approximately 70% of alveolar RMSs harbor t(2;13) or t(1;13) chromosomal translocations that result in PAX3-FOXO1 or PAX7-FOXO1 oncoprotein expression.In particular, PAX3-FOXO1 may be a key biomarker patients' risk-stratification being correlated to the poorest outcome [3].Despite improvement in multimodality treatments for high risk RMS, the management of those patients remains challenging, with a 5-year overall survival less than 30%.Therefore, understanding the molecular pathways that contribute to the pathogenesis and self-propagation of the most aggressive tumor forms is urgently needed. RMS cells express key myogenic factors such as MyoD and Myogenin, but proliferate indefinitely and have lost the ability to terminally differentiate into skeletal myofibers [4]. The Notch signaling pathway plays fundamental roles in balancing proliferation versus differentiation [5] and is one of the major regulators of skeletal muscle tissue development.Mammals harbor four Notch genes, each encoding a type I trans-membrane Notch receptor paralog (Notch1-4).Notch receptors are most commonly activated after binding to the extracellular domain of a trans-membrane ligand of Delta-like (DLL1, DLL3-4) or Serrate/Jagged (JAG1-2) family on neighboring cells.The Notch-ligand interaction allows Notch to undergo sequential proteolytic cleavages, the last one being mediated by the c-secretase complex that releases an active Notch intracellular domain (NotchIC).NotchIC translocates into the nucleus, where it behaves as a transcriptional regulator in complex with the DNA-binding RBP-Jk protein (also known as CSL/RBP-Jk, for CBF1/Su(H)/Lag1) inducing the expression of target genes [6].Among canonical Notch target genes are those encoding the Enhancer of split group of transcriptional repressors, which are termed Hairy and Enhancer of split (HES) 1-7 and HES-related repressor (HEY) 1,2 and L in mammals [7]. In skeletal muscle progenitors, Notch1 activation impairs the transcription of myogenic regulatory factors, promoting proliferation and self-renewal of myogenic precursors [8,9,10,11,12].Notch3 expression induces de-differentiation of myoblasts and, more recently, it has been shown to prevent myogenic differentiation by affecting Mef2c activity [13].Consistent with these observations, inhibition of either c-secretase activity or RBP-Jkdependent gene transcription leads to myotube fusion [14,15,16]. Recently, we and others have shown that Notch signaling is deregulated in RMS [17,18,19,20,21].General inhibition of Notch signaling with different approaches inhibits the proliferation of RMS cells [20] and prevents their migration and invasion [18].Interestingly, the inhibition of the Notch1-HEY1 axis specifically impaired the proliferation of embryonal RMS cells, but it had only marginal effects on their differentiation properties [21].Recently, we have shown that Notch3 prevented the differentiation of both subtypes of RMS cells [19].Consistent with the data of Sang et al. [17], this function was, at least in part, related to the Notch3dependent induction of HES1.We also reported that Notch3 inhibition hampered the growth of PAX3-FOXO1 alveolar RMS cells in vivo [19].In addition, we observed that, in line with previous results [21], Notch1 knockdown was not sufficient to induce the differentiation of RMS cells, irrespective of their subtype [19].These results are consistent with data showing that (i) myogenic differentiation and proliferation, though coupled, can be considered independent events during myogenesis [22] and (ii) different Notch paralogs can have different and even opposite roles in the same cellular/tissue context [23,24,25,26,27]. Therefore, we investigated the role of Notch3 activation by forcing the expression of an exogenous Notch3IC in RMS cells.We asked whether, in addition to regulating the differentiation of RMS cells, Notch3 may also play a role in sustaining their proliferation.Moreover, the relationship between Notch3 and/or HES1 expression and the proliferative status of primary RMS samples were evaluated. We show here that Notch3IC over-expression in two PAX3-FOXO1-positive alveolar and one PAX3-FOXO1-negative embryonal RMS cell lines further increased their proliferative activity in vitro.Moreover, PAX3-FOXO1 alveolar cells over-expressing Notch3IC grew faster when xenografted into nude mice.Notably, forced over-expression of Notch3IC prevented the anti-proliferative effects of a c-secretase inhibitor (GSI), suggesting that the inhibitory effect of this drug was mediated at least in part by Notch3 inhibition.We also expand our previous data showing that Notch3 signaling in RMS cells is not hyper-activated by a cellautonomous mechanism, as reported in some other cancers [28,29,30,31], but depends largely upon both types of canonical ligands.Finally, we report preliminary data showing that Notch3 activation and HES1 expression were highest in primary PAX-FOXO1-positive alveolar RMS samples with the highest levels of proliferative marker Ki67.Altogether, our results support a role for Notch3 activation as an amplifier of the proliferation potential of RMS cells and suggest that selected alveolar RMS may clinically benefit from either Notch inhibition or Notch ligand blockade ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "Tumor cell lines RH30 (PAX3-FOXO1 expressing alveolar RMS) and RD (embrional RMS) cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).RH41 (PAX3-FOXO1 expressing alveolar RMS) cell line was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany).RH30 and RH41 cells were cultured in RPMI 1640 (Invitrogen Corp., Carlsbad, CA, USA) while RD cells were cultured in DMEM (Invitrogen Corp., Carlsbad, CA, USA) supplemented with 10% FCS, 1% glutamine and 1% penicillin-streptomycin at 37uC in a humidified atmosphere of 5%CO2/95% air.Several first passage aliquots of each cell line were stored in liquid nitrogen at 280uC for subsequent assays.Each aliquot was passaged for a maximum of 3 months. ",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "For c-secretase inhibitor treatment, 24 h after seeding (day0) in 6-well plates (56104 cells/well) cells were treated with 5 mM of N-[N-(3,5-Difluoro-phenacetyl)-L-alanyl]-S-phenylglycine t-butyl Ester (DAPT) (Sigma, St Louis, MO, USA) in DMSO (10 mmol\\L stock solution).Cells treated with DMSO alone (vehicle) were used as control.Cells were harvested at different time points and living cells were counted with the trypan blue exclusion method. ",
"section_name": "Cell proliferation",
"section_num": null
},
{
"section_content": "Western blotting was performed on whole-cell lysates as previously described [32].Antibodies against Notch1 (all forms; bTAN20) and Myogenin (F5D) were obtained from the Developmental Studies Hybridoma Bank at the University of Iowa (DSHB, Iowa City, IA, USA).Notch3 antibody (PAB-10683) was obtained from Orbigen Inc. (Orbigen, San Diego, CA, USA).Antibodies against Notch1-3 recognize an intracellular region within the c-secretase cleavage product of each Notch molecule i.e., NotchIC.Antibodies against HES1 (sc-25392), p21Cip1 (sc-397), actin (sc-1616) and all secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).Antibodies against Phospho-Akt (Ser473), Akt (pan) (C67E7), Phospho-p38 (Thr180/Tyr182), p38 MAP Kinase, Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), p44/42 MAPK (ERK1/2) (137F5), Jagged1 (28H8), DLL1 (2588) and GAPDH (D16H11) were obtained from Cell Signaling (Cell Signaling Technology Inc., Beverly, MA, USA).Antibody against tubulin (ab4074) was from Abcam (Abcam PLC., Cambridge, UK).All the antibodies were used in accordance with the manufacturer's instructions. ",
"section_name": "Western blotting",
"section_num": null
},
{
"section_content": "Total RNA was extracted from RH30 cells using Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA), according to the manufacturer's instructions.For the relative quantification of HES1 gene expression the TaqMan gene assay Hs00172878_m1 was used according to the manufacturer's instructions (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). ",
"section_name": "Real time RT-PCR",
"section_num": null
},
{
"section_content": "The plasmids pcDNA3.0\\Notch3ICexpressing the intracellular domain of human Notch3 (Notch3IC) (M.Bocchetta, Loyola University, Chicago, MC, USA) or the empty pcDNA3.0 were transfected using Lipofectamine 2000 (Invitrogen Corp., Carlsbad, CA, USA).Cells were selected with G418 (1.5 mg/ml) (4 weeks RH30 and RD and 1 week RH41 cells) and, then, used for the experiments. ",
"section_name": "Plasmids and transfection",
"section_num": null
},
{
"section_content": "Stably transfected Notch3IC or control vector RH30 cells were assayed for their capacity to form colonies in soft-agar as previously described [32].Briefly, a total of 30000 cells was suspended in DMEM (10% FCS) containing 0.35% Noble agar (Sigma Chemical Co., St Louis, MO, USA).Cells were seeded on a layer of 0.7% Noble agar in DMEM (10% FCS) onto a 35-mm Petri dish.Medium was refreshed every 5 days.On week 4, the number of colonies per field was counted under the contrast-phase Eclipse TE200 microscope (Nikon, Sesto Fiorentino, Firenze, Italy).Two independent experiments were carried out in triplicate. ",
"section_name": "Soft-agar colony formation assay",
"section_num": null
},
{
"section_content": "Athymic 6-week-old female BALB/c nude mice (nu+\\nu+) were purchased from Charles River.Procedures involving animals and their care conformed to institutional guidelines that comply with national and international laws and policies (EEC Council Directive 86\\609, OJ L 358, 12 December 1987).The protocol was approved by the Committee on the Ethics of Animal Experiments of the Italian Ministry of Health to the ''Stabilimento Allevatore, Fornitore Utilizzatore'' S.A.F.U., IFO, Roma (4/03/ 2010).All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.RH30 cells (2610 6 ) stably transfected with either pcDNA3.0\\-Notch3IC(RH30-Notch3IC) or pcDNA3.0vector (RH30-Vector) were injected subcutaneously into the posterior legs of nude mice in a cold mixture of PBS\\Matrigel (ratio 1:1).When tumors became palpable, about 15 days after the initial inoculation, their growth was monitored and measured twice weekly by caliper.Tumor volume was calculated with the following formula: tumor volume (mm 3 ) = L6S 2 6p/6 wherein L is the longest and S the shorter diameter and p/6 is a constant to calculate the volume of an ellipsoid.Relative volume was plotted against time in days to determine tumor growth.Representative tumor growth data were obtained from at least 5 mice per experimental group. In a second set of experiments, animals were sacrificed at specific time points, the tumors were excised and portions were embedded in paraffin and snap-frozen in OCT for immunohistochemical analysis.Additionally, portions were frozen in liquid nitrogen to analyze Notch3IC expression by Western blot.Ten mm sections cut from xenograft blocks were stained with hematoxylin/eosin.Five mm sections were immunostained for Ki67 and HES1 with antibodies and methods as described for primary human RMS samples in Table S2.Counterstaining was carried out with Gill's hematoxylin (Bio-Optica, Milan, Italy).Sections were dehydrated and mounted in non-aqueous mounting medium.Images were acquired through an Eclipse E600 microscope (Nikon, Sesto Fiorentino, Firenze, Italy) at 400X Magnification.Sections were scored using LUCIA software, version 4.81 (Nikon, Sesto Fiorentino, Firenze, Italy) with a Nikon Digital Camera DXM1200F. ",
"section_name": "Xenograft growth",
"section_num": null
},
{
"section_content": "Cells were transfected with 100 nM (final concentration) of double-stranded synthetic 21-mer RNA oligonucleotides (siRNA) using Oligofectamine (Invitrogen, Carlsbad, CA, USA).The most effective JAG1 (NM_000214.2) and DLL1 (NM_005618.3)siRNA, chosen among three different tested Silencer Select siRNAs (Applied biosystems, CA, USA/Ambion, Life Technologies Italia, Monza (MI), Italy) are referenced as s1176 and s26277, respectively.The non-targeting Silencer Select Control N. 1 was the control siRNA (Applied Biosystems, CA, USA/Ambion, Life Technologies Italia, Monza (MI), Italy).siRNA effectiveness was validated by Western blotting and RT-PCR (data not shown). ",
"section_name": "RNA interference",
"section_num": null
},
{
"section_content": "Archival, de-identified formalin-fixed, paraffin-embedded primary RMS and skeletal muscle control tissues were obtained from the Ospedale Pediatrico Bambino Gesu `in Roma, University of Padova and Fondazione IRCCS Istituto Tumori of Milano (Italy) after approval of the respective Ethical Committees (EC of Ospedale Pediatrico Bambino Gesu `, Roma; EC of the Fondazione IRCCS Istituto Nazionale dei Tumori, Milano; EC of University Hospital of Padova, Padova).We confirm that written informed consent from each donor or parent/guardian when applicable was obtained for use of these samples in research. ",
"section_name": "Ethics statement",
"section_num": null
},
{
"section_content": "Clinicopathological characteristics of the cohort are reported in Table S1.Histopathological features of the tumors were reviewed for the present study by a Pathologist of each Institution (R.B., R.A and P.C) blinded to the results of immunohistochemical analysis.All alveolar tumor samples harbored either PAX3-FOXO or PAX7-FOXO fusion transcripts.Embryonal samples were negative for these gene fusions.Marker expression was evaluated in sections from samples with sufficient available material.Immunohistochemistry was performed with antibodies and immunohistochemistry conditions reported in Table S2.Sections were scored by two independent observers blinded to the identity of the samples and, in rare cases of discrepancy, by an additional third independent observer.Marker positivity was determined on the basis of discrete nuclear staining and a semi-quantitative score of the percentage of positive stained cells/field as the average obtained by scoring at least 5 fields per section.Images were acquired through an Eclipse E600 microscope (Nikon, Sesto Fiorentino, Firenze, Italy) at 400X Magnification.Sections were scored using LUCIA software, version 4.81 (Nikon, Sesto Fiorentino, Firenze, Italy) with a Nikon Digital Camera DXM1200F. ",
"section_name": "Clinical specimens and immunohistochemistry",
"section_num": null
},
{
"section_content": "",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To explore the impact of Notch3 activation on the proliferative potential of RMS cells, we transfected the PAX3-FOXO1-positive alveolar RH30 and PAX3-FOXO1-negative embryonal RD cell lines with a plasmid over-expressing an exogenous cleaved intracellular (active) domain of Notch3, i.e.Notch3IC.Stably Notch3IC-over-expressing RH30 and RD cells showed an increased rate of proliferation, evident as early as 3 days post-seeding.At day 6 post-seeding, Notch3IC cell number had increased by 8.5 fold (RH30 cells) and 7.8 fold (RD cells) compared to 5.2 fold and 4.5 fold for control vector-transfected cells, respectively (Figure 1A).This phenomenon was associated to HES1 protein level up-regulation as compared to empty vectortransfected control cells (Figure 1B).Consistently, both RH30-and RD-Notch3IC cells displayed higher levels of ERK1/2 phosphorylation (Figure 1B).In line with previous results on Notch3 depletion and with the anti-differentiation role of Notch3 in the same cell lines [19], Notch3IC over-expression decreased the levels of both cyclin-dependent kinase inhibitor p21Cip1 and Myogenin along with the activation/phosphorylation of p38 MAPK (mitogen-activated protein kinase) and serine-threonine kinase Akt, all essential for terminal muscle differentiation (Figure 1B).Similar results were obtained with an additional, commercially available PAX3-FOXO1-positive alveolar RMS cell line (RH41) transiently transfected with Notch3IC or control vector (Figure S1A andB).Of note, Notch3IC over-expression also markedly increased the ability of RH30 and RD cells to form colonies in soft-agar as compared to control vector (Figure 1C). ",
"section_name": "Forced expression of a cleaved form of Notch3 (Notch3IC) increases cell proliferation and soft-agar colony formation in vitro of RMS cell lines",
"section_num": null
},
{
"section_content": "Next, we decided to explore the effect of Notch3IC overactivation on the in vivo growth potential of the aggressive PAX3-FOXO1 alveolar RH30 cell line.RH30-Notch3IC or control RH30-Vector cells were subcutaneously inoculated in immunocompromised mice and the growth of xenografted tumors was monitored.A significant difference in tumor volume between the two groups of animals was detected by the 4th day after the first measurement (see Methods), with Notch3IC-expressing cells consistently giving rise to larger tumors (Figure 2A).The sustained over-expression of Notch3IC was verified by Western blotting in tumor xenografts excised at days 16 and 34 (Figure 2B).Consistent with an increased proliferative capability, Ki67 expressing cells were more numerous in xenografts derived from RH30-Notch3IC cells.RH30-Notch3IC cells also expressed higher levels of Notch3 target gene HES1 compared to RH30-Vector cells (Fig 3A andB). Altogether, these results indicate that Notch3 hyper-activation amplifies the proliferative potential of RMS cells in vitro and in vivo. ",
"section_name": "Notch3IC increases the in vivo tumorigenic potential of RH30 cells",
"section_num": null
},
{
"section_content": "Inhibitors of c-secretase (GSIs) non-selectively block the activation of Notch signaling in many cell types [33].GSIs block the final protelolytic cleavage that releases the Notch intracellular domains (and those of other transmembrane proteins).The antiproliferative effect of GSIs has been reported for embryonal RMS cell lines [20,21].Here, we examined the effect of GSI N-[N-(3,5-Difluoro-phenacetyl)-L-alanyl]-S-phenylglycine t-butyl Ester (DAPT) on the proliferation of RH30 and RD cells cultured in a pro-proliferative medium (i.e., supplemented with 10% FCS).Five days of treatment with 5 mM DAPT decreased proliferation by 60% in RH30 cells compared to vehicle (Figure 4A).A smaller but still significant effect (34%) was seen in RD cells.These findings were associated with significant decrease of HES1 transcripts and protein levels 3 days post-treatment in RH30 and RD cells, respectively, confirming the effectiveness of DAPT in blocking canonical Notch signaling (Figure 4B andC).RH41 cells were also sensitive to DAPT and showed HES1 downregulation (Figure S2A, B andC). Similar results were obtained on the RH30 and RD cells with a chemically distinct c-secretase inhibitor, GSI XII (Figure S3).A marked decrease of the levels of Notch3IC associated to an increase of uncleaved receptor (Notch3FL) was observed after 3 days of DAPT treatment compared to vehicle controls for all the cell lines tested, confirming inhibition of Notch3 cleavage (Figure 4C and Figure S2C).Interestingly, a decrease in Notch1IC accompanied by a modest increase in Notch1FL was observed in RH30 and RH41 cells treated with DAPT, while this effect was more marked in RD cells (Figure 4C and Figure S2C). To assess whether forced expression of Notch3IC could overcome the anti-proliferative effect of DAPT, we performed a rescue experiment treating Notch3IC cells with the GSI.Treatment with DAPT significantly decreased cell proliferation in empty vector-transfected RMS cells as early as 4 days after treatment (Figure 4D and E, and Figure S2D), as reported for wild-type cells.Conversely, all the Notch3IC-over-expressing RMS cell lines were insensitive to DAPT (Figure 4D andE, and Figure S2D).These findings suggest that forcing Notch3 activation increases RMS cell proliferation and renders cells GSI-resistant, irrespective of their fusion oncoprotein expression or the inhibition of other Notch paralogs. ",
"section_name": "Notch3IC over-expression overcomes the antiproliferative effect of c-secretase inhibitor DAPT in RMS cells",
"section_num": null
},
{
"section_content": "Hyper-activation of Notch receptors in pediatric leukemias is often, though not always, due to mutations of Notch genes that allow ligand-independent cleavage [28,29,30,31].This is rarely true for solid cancers, in which very few Notch-activating mutations have been described [34,35].We previously showed that Notch3 cleavage is partly dependent on binding to the Serrate/Jagged family Notch canonical ligand JAG1 [19].Having detected the expression of the Delta-like Notch canonical ligand DLL1 in RMS cells (data not shown), we evaluated whether DLL1 as well contributes to Notch3 activation in these cells.As shown in Figure 5A, a marked down-regulation of the Notch3IC form was detected not only after JAG1 but also after DLL1 knockdown by specific siRNAs in RH30 and RD cells compared to cells transfected with non-targeting control (CTR) siRNAs.RH41 cells behaved similarly (Figure S4A).To elucidate the functional effects of ligand knockdown, we investigated the proliferative potential of siRNAs-treated cells.A significant anti-proliferative response was seen after either JAG1 or DLL1 knockdown in both RH30 and RD cells as early as 3 days after treatment and was maintained up to 4 days (Figure 5B).This effect, though still present 4 days posttreatment, was delayed in RH41 cells (Figure S4B).Therefore, the activation of Notch3 in RMS cells is dependent, at least in part, on canonical ligands. ",
"section_name": "The hyper-activation of endogenous Notch3 in RMS cells is ligand-dependent",
"section_num": null
},
{
"section_content": "The transcript levels of Notch1 and Notch3, together with their respective target genes in RMS, i.e.HEY1 and HES1, in RMS primary samples have been examined by several groups [18,21,36].Therefore, here we evaluated the nuclear expression of the two Notch receptors and of their direct target genes.To this end, we performed immunohistochemical analyses on primary RMS samples.Due to the evidence that fusion-negative alveolar tumors seem to be molecularly and clinically indistinguishable from embryonal ones [37], we examined PAX3-FOXO1-(n = 10) and PAX7-FOXO1-positive (n = 2) alveolar (n = 12 in total) and fusion-negative embryonal (n = 20) RMS samples (Table S1).A well defined nuclear expression, i.e. the presence of the transcriptionally active form of the receptor, was considered to be a marker of Notch receptor activation.We detected higher nuclear expression of Notch3 and HES1 (Figure 6), Notch1 and HEY1 (Figure 7) along with that of Ki67 (Figure 8) in RMS samples compared to control skeletal muscle tissues whose myofibers were negative for all markers (Table 1). No significant difference was noticed in the numbers of nuclei expressing either Notch3 or HES1 between PAX-FOXO1 alveolar and embryonal RMS samples (Table 1).Instead, consistent with a previous report despite somewhat lower numbers [21], the fractions of nuclei positive for Notch1 and HEY1 were higher in the embryonal subset.However, in PAX-FOXO1positive patients, Notch3-and HES1-stained nuclei were significantly more numerous than nuclei positive for Notch1 and HEY1 (Table 1).These results suggest that the Notch3-HES1 axis may be a major Notch-activated signaling pathway in PAX-FOXO1positive RMS.Moreover, Notch3 and HES1 levels directly correlated with Ki67 in this subgroup (r = 0.676, P = 0.016 and r = 0.644, P = 0.024, respectively). In summary, our data suggest that Notch3 promotes the proliferation of RMS cells in vitro and in vivo and that a Notch3-HES1 axis may support the proliferation of some pediatric RMS. ",
"section_name": "Notch3 is activated and HES1 protein levels up-regulated in RMS primary samples",
"section_num": null
},
{
"section_content": "We previously demonstrated that Notch3 activation, in part through the expression of its target gene HES1, prevents the in vitro differentiation of both embryonal and PAX3-FOXO1-positive alveolar RMS cell lines and sustains the in vivo growth of PAX3- FOXO1 alveolar cells [19].We showed that Notch3 knockdown is sufficient to reverse these tumorigenic features [19]. Here, we provide evidence that forced over-expression of exogenous Notch3 activated domain, Notch3IC, further stimulates cell proliferation and anchorage-independent growth of both PAX3-FOXO1 alveolar and embryonal RMS cells in vitro, even in medium supplemented with 10% FCS that strongly supports the proliferation of RMS cells.Our observations are consistent with data of Nagao et al. on embryonal RMS RD cells over-expressing an exogenous RBP-Jk protein, suggesting that forcing Notch signaling activation can enhance the intrinsic pro-tumorigenic characteristics of RMS cells [20].Notch3IC cells expressed higher levels of ERK1/2 phosphorylation and lower levels of p21Cip1 as compared to control vector cells, consistent with stimulation of cell cycle progression.The concomitant down-regulation of Myogenin, Akt and p38 MAPK indicates a concomitant anti-differentiation effect. Importantly, GSI-mediated inhibition of Notch signaling strongly reduced cell proliferation in both RMS cell subtypes.GSI DAPT markedly affected the cleavage of Notch3 and the expression of its target gene HES1, suggesting endogenously high activity of this Notch paralog in RMS cells.Consistent with this observation, Notch3IC over-expression completely prevented the anti-proliferative effects of GSI in all RMS cell lines, ruling out a cell line-or cell subtype-dependent effect.Even though these results may suggest that c-secretase inhibition has a Notch3targeted effect in RMS cells, recent results revealed a similar response when exogenous Notch1IC was expressed in embryonal RD cells [21].Results from Linardic' group [21] and our data support the hypothesis that hyper-activation of either Notch1 or Notch3 signaling is sufficient to rescue the effects of GSI, suggesting that: 1) these Notch receptors converge on the same proliferative nodal points, at least in embryonal RMS cells and 2) when expressed in sufficient amounts, Notch1IC and Notch3IC The lower pictures depict selected regions at higher magnification (600X) for each marker.Black and red arrows indicate nuclei either positive (brown) or negative (blue-gray) for each marker, respectively.Left panels report the immunohistochemistry for each marker in normal skeletal muscle used as control tissue, in which myofibers' nuclei are negative for the two markers.Yellow arrows: vessels with vascular smooth muscle cells positive for Notch3 expression (orange staining).doi:10.1371/journal.pone.0096238.g006can be functionally redundant in these cells.Further studies may elucidate whether this is also true for PAX3-FOXO1 RMS cells. RH30-Notch3IC cell inoculation in nude mice resulted in the formation of larger tumors compared to vector control cells.Notch3IC expression was maintained in these tumors.Since excessive Notch signaling activity is often toxic for several cell types [38], this result suggests that Notch3 hyper-activation is well tolerated in PAX3-FOXO1-harboring RMS cells and supports tumor growth. JAG1 and DLL1 knockdown experiments indicate that in the RMS cell lines we evaluated, Notch3 activation is liganddependent and both JAG family and DLL family ligands can trigger Notch3 activation.Ligand-independent activation has been described in other tumor cell types [28,29,30,31].Silencing of either JAG1 or DLL1 mirrored the anti-proliferative effect of GSI, although the effect size was markedly lower.Whether this difference is due to incomplete ligand knockdown or to the fact that both ligand families contribute to Notch3 activation in these cells remains unclear.However, collectively, our data strongly support a pro-proliferative role for Notch3 signaling in RMS cells. Roma et al. [18] first showed de-regulation of Notch signaling in RMS patients, quantifying transcript levels for several Notch components, including the ones reported in this study.Subsequently, the Linardic group analyzed the nuclear protein expression of Notch1 and its target gene HEY1, as surrogates of Notch1 signaling activation, in primary RMS samples [21].Here, we performed a similar pilot study to assess Notch3 signaling activation levels.Our findings show that Notch3 is activated and HES1 over-expressed in primary samples from RMS patients, irrespective to their fusion status, compared to normal skeletal muscle tissue.Interestingly, even though the staining fraction was lower in our cohort compared to previous reports [21], Notch1 and HEY1 nuclear expression were significantly higher in embryonal RMS samples.However, in PAX-FOXO1-positive The lower pictures depict selected regions at higher magnification (600X) for each marker.Black and red arrows indicate nuclei either positive (brown) or negative (blue-violet) for each marker, respectively.Green arrows: Notch1 cytoplasmic staining.Left panels report the immunohistochemistry for each marker in normal skeletal muscle used as control tissue, in which myofibers' nuclei are negative for the two markers.A Notch1-positive nucleus of vessel cell was highlighted with black arrow.doi:10.1371/journal.pone.0096238.g007alveolar RMS samples, nuclear levels of Notch3 and HES1 were markedly higher than those of Notch1 and HEY1.This finding suggests that the Notch3-HES1 axis could be a major driver in alveolar RMS bearing PAX3-FOXO1 translocations in vivo.We also noticed that the levels of Notch3 and HES1 were both correlated with proliferation markers in these tumors, suggesting that a hyper-activated Notch3-HES1 axis may contribute to tumor aggressiveness in vivo [3].However, given that Notch3 hyperactivation showed a similar effect in both tumor cell subtypes, we can argue that the proliferative effects of Notch3IC do not absolutely require expression of the PAX3-FOXO1 fusion oncoprotein.This hypothesis is corroborated by our previous results showing that Notch3 knockdown is detrimental for cells of both RMS subtypes [19], and by data from the Helman group demonstrating that Notch signaling is not among the molecular pathways regulated by PAX3-FOXO1 in RH30 and RH4 RMS cell lines [39].However, the evidence that Notch3 correlates with Ki67 only in PAX3-FOXO1-positive RMS suggests that simultaneous activation of the Notch3 and PAX3-FOXO1 pathways is a common occurrence in this subset of RMS [37,40], highlighting once more the difference between the RMS subtypes [41,42].In any case, further studies are needed to understand whether and how Notch3 and PAX3-FOXO1 crosstalk or converge on common pathways in RMS. In conclusion, these results support a role for Notch3 as an amplifier of the proliferative features of RMS cells and suggest that therapeutic targeting of Notch3 may potentiate the effects of conventional therapy in pediatric RMS.Values normalized to actin levels were expressed as fold increase over vehicle-treated (1 arbitrary unit).Two independent measurements were done in duplicate.C, Western blotting showing the expression of full length Notch3 (Notch3FL), Notch3IC, Notch1FL, Notch1IC along with that of HES1, 72 h after DAPT treatment.Tubulin was the loading control.D, RH41 cells were transiently transfected with either a pcDNA3 plasmid expressing Notch3IC (Notch3IC) or with an empty pcDNA3 plasmid as control vector (Vector).Seventy-two hours after transfection polyclonal cell populations were treated with G418 for 1 week and, then, seeded in a 6wells plate, treated with DAPT (5 mM) or vehicle (DMSO) and then harvested and counted at the reported time points.Representative of two independent experiments in triplicate (*P,0.05);Bars, SD. (JPG) ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "We wish to thank the Animal Facility team of SAFU, Ifo, Roma, Italy. ",
"section_name": "Acknowledgments",
"section_num": null
}
] |
10.1038/s41598-018-35252-3
|
DLK proteins modulate NOTCH signaling to influence a brown or white 3T3-L1 adipocyte fate
|
<jats:title>Abstract</jats:title><jats:p>The role of NOTCH signaling in adipogenesis is highly controversial, with data indicating null, positive or negative effects on this differentiation process. We hypothesize that these contradictory results could be due to the different global NOTCH signaling levels obtained in different experimental settings, because of a specific modulation of NOTCH receptors’ activity by their ligands. We have previously demonstrated that DLK1 and DLK2, two non-canonical NOTCH1 ligands that inhibit NOTCH1 signaling in a dose-dependent manner, modulate the adipogenesis process of 3T3-L1 preadipocytes. In this work, we show that over-expression of any of the four NOTCH receptors enhanced adipogenesis of 3T3-L1 preadipocytes. We also determine that DLK proteins inhibit not only the activity of NOTCH1, but also the activity of NOTCH2, 3 and 4 receptors to different degrees. Interestingly, we have observed, by different approaches, that NOTCH1 over-expression seems to stimulate the differentiation of 3T3-L1 cells towards a brown-like adipocyte phenotype, whereas cells over-expressing NOTCH2, 3 or 4 receptors or DLK proteins would rather differentiate towards a white-like adipocyte phenotype. Finally, our data also demonstrate a complex feed-back mechanism involving <jats:italic>Notch</jats:italic> and <jats:italic>Dlk</jats:italic> genes in the regulation of their expression, which suggest that a precise level of global NOTCH expression and NOTCH-dependent transcriptional activity of specific targets could be necessary to determine the final phenotype of 3T3-L1 adipocytes.</jats:p>
|
[
{
"section_content": "transmembrane proteins with six extracellular EGF-like repeats that interact with the NOTCH1 receptor and function as NOTCH signaling inhibitors [20][21][22] .DLK1 and DLK2 lack a DSL domain, although both possess a DOS domain that is postulated to function by competing with the canonical NOTCH receptor ligands 23 . Accumulated evidence indicates that Dlk1 is involved in several cell differentiation processes, including adipogenesis.Thus, several works, some of them performed in Dlk1-deficient and transgenic mice, point to Dlk1 as an inhibitor of adipogenesis 22,[24][25][26][27][28] .Several research groups, including ours, have demonstrated that DLK1 over-expression inhibits 3T3-L1 adipogenesis, whereas enforced decrease in Dlk1 expression enhanced this differentiation process.Our research group furthermore demonstrated that Dlk2 also modulates adipogenesis of 3T3-L1 cells 17 .On the other hand, recent studies implicate Dlk1 in the control of whole body metabolism 29,30 , the onset of diabetes in humans 31,32 , and adipocyte browning 33,34 . Several mechanisms have been proposed to explain the action of DLK proteins on 3T3-L1 adipogenesis [35][36][37][38][39][40][41] .However, probably the most important fact revealed by our and other research groups is that both proteins interact with the NOTCH1 receptor and function as inhibitory non-canonical ligands of NOTCH1 signaling in a dose-dependent manner 7,21,[35][36][37][38][39][40][41][42][43] .We then hypothesized that these proteins could regulate adipogenesis and adipocyte phenotype by generating defined levels of NOTCH1 signaling, leading or not to the progression of this differentiation process.However, in our view, to achieve this precise level of NOTCH signaling, DLK proteins should not only modulate NOTCH1 activity, but also the activity of the other three NOTCH receptors. In this work, we show that over-expression of any of the four NOTCH receptors enhanced the adipogenic potential of 3T3-L1 preadipocytes, and that DLK proteins can inhibit the activity of NOTCH1, 2, 3 and 4 receptors to different degrees.Interestingly, we have demonstrated by performing different assays that NOTCH1 over-expression drives differentiation of 3T3-L1 cells towards a brown-like adipocyte phenotype, whereas preadipocytes over-expressing NOTCH2, 3 or 4 receptors or DLK proteins develop a gene expression profile and phenotype more similar to white adipocytes.Finally, we also observed that over-expression of any of the four NOTCH receptors and their signaling and the over-expression of any DLK protein affect the expression levels of the others to different degrees.To sum up, all these data suggest the existence of a complex feedback regulation mechanism involving the expression of all Notch and Dlk genes that may lead to a precise level of NOTCH signaling to allow 3T3-L1 preadipocytes to differentiate or not to a particular adipocyte phenotype. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "The role of NOTCH signaling in the adipogenesis process is a highly controversial topic.Rather than attributing the contradictory results to technical problems or cell line differences, we hypothesized that they could be related to the possibility that adipogenesis in response to extracellular signals may proceed or not given a precise level of global NOTCH receptor activation 22 . We first analyzed here the basal mRNA expression levels of Notch genes in 3T3-L1 cells and found that these cells express similar levels of the four Notch genes (Supplementary Figure 1A).We observed that the induction of adipogenesis in 3T3-L1 cells increased aP2 and Pparg mRNA expression (Fig. 1A).In addition, whereas the expression of Notch1, Notch2, Notch3, and Hes1 mRNAs increased with the adipogenic treatment, the expression of Notch4 mRNA decreased (Fig. 1B).Western blot analysis also demonstrated that NOTCH4 protein expression decrease at the end of the adipogenic process, whereas the expression of NOTCH1, 2 and 3 increases (Fig. 1C,D). Next, pools of 3T3-L1 preadipocytes stably over-expressing each one of the Notch genes were used to investigate the effects of each NOTCH receptor on 3T3-L1 adipogenesis.The over-expression of Notch genes and their proteins was confirmed by qRT-PCR and Western blot analysis (Supplementary Figure 1(B-I)).We have observed that over-expression of any Notch gene enhanced the adipogenic potential of these cells, as indicated by an increase in aP2 expression (Fig. 1E).Pparg expression also increased in cells over-expressing Notch1 gene, but its levels drastically decreased in differentiated cells that over-expressed Notch2, Notch3 and Notch4 genes (Fig. 1E). ",
"section_name": "Over-expression of each one of the NOTCH receptors enhances adipogenesis of 3T3-L1 preadipocytes.",
"section_num": null
},
{
"section_content": "cytes have been always referred to as a typical cellular model of white adipogenesis.However, some authors described that 3T3-L1 adipocytes display phenotypic characteristics of multiple adipocyte lineages 44 .In this work, we have observed that the induction of adipogenesis in 3T3-L1 cells generates adipocytes (L1D) with multilocular lipid droplets (Fig. 2A), and increases the expression of the brown adipocyte and mitochondrial biogenesis markers Pgc1a, Ucp1, Gyk and Prdm16 (Fig. 2B).We also observed a decrease in Cidea expression, which might be associated with the multilocular droplets observed 45 .However, the expression of the mitochondrial biogenesis marker Sirt1 decreased in 3T3-L1 differentiated cells (Fig. 2B). Taking these results into consideration, we were interested in exploring whether the stable over-expression of each one of the four NOTCH receptors could also affect the expression levels of the brown adipocyte genes after induction of adipogenesis (Fig. 2).Compared with differentiated control cells, Notch1 over-expressing adipocytes showed higher expression levels of the brown adipocyte and the mitochondrial biogenesis markers Pgc1a, Ucp1, Gyk, Prdm16, Cidea and Sirt1 (Fig. 2C).Likewise, Notch2 over-expression diminished Pgc1a, Ucp1, Gyk, and Cidea expression, although no significant differences were observed in Prdm16 and Sirt1 expression (Fig. 2D).Notch3 over-expression diminished the expression of Ucp1, Gyk, Prdm16 and Sirt1 in differentiated cells, although Pgc1a levels were increased and no significant differences were observed in Cidea expression (Fig. 2E).Finally, Notch4 over-expression diminished the expression levels of Pgc1a, Ucp and Gyk in differentiated cells, but it increased the expression of Prdm16, Cidea and Sirt1 (Fig. 2F).We also analyzed the amplification levels of mitochondrial CytB gene in these differentiated stable transfectants (see Methods), which indicates the rate of mitochondrial biogenesis.The over-expression of Notch1 increases the amplification level of mitochondrial CytB gene in differentiated cells (Fig. 2G), whereas the over-expression of Notch4 decreases the amplification of this gene (Fig. 2H).No significant differences in mitochondrial CytB gene amplification were found when cells over-express Notch2 or Notch3 genes (Fig. 2H). Being inhibitors of NOTCH signaling, we expected that stable over-expression of Dlk1 or Dlk2 would affect also the expression of these markers (Fig. 3).Thus, we generated 3T3-L1 cells over-expressing Dlk1 or Dlk2 (Supplementary Figure 2) to study this hypothesis.We observed that Dlk1 over-expression significantly increased the expression of Pgc1a and Gyk, decreased that of Cidea and Sirt1, and left that of Ucp1 and Prdm16 unaffected (Fig. 3A).On the other hand, cells stably over-expressing Dlk2 showed a decrease in Gyk, Cidea, Sirt1 expression, although no significant differences were observed in Pgc1a, Prdm16 and Ucp1 expression (Fig. 3A).We also analyzed the amplification levels of mitochondrial CytB gene amplification in these stable transfectants (see Methods).The over-expression of Dlk2 decreases the amplification levels of this gene (Fig. 3B).No significant differences in mitochondrial CytB gene amplification were found when cells over-express Dlk1 gene (Fig. 3B). To estimate the cytoplasmic lipid accumulation, we measured the amount of glycerol released from the different transfected adipocytes by inducing lipolysis with the β-adrenergic agonist isoproterenol (Fig. 4).3T3-L1 transfectants over-expressing Dlk1 released lower glycerol levels into the medium than control cells (Fig. 4A), qRT-PCR analysis of the relative aP2 and Pparg mRNA expression levels in differentiated stable Notch1 gene transfectant (L1-N1D), stable Notch2 gene transfectant (L1-N2D), stable Notch3 gene transfectant (L1-N3D), and stable gene Notch4 transfectant (L1-N4D).Data from qRT-PCR assays were previously normalized to P0 mRNA expression levels.The expression of alpha-tubulin was used as a loading control in all Western blots to normalize expression data.Blot signals from empty vector and over-expressing cells were cropped from original blots and delineated with horizontal white spaces (original blots for each protein signal are shown in Supplementary Figure 4).The fold activation or inhibition was calculated relative to the seven-day differentiated non-transfected or empty-vector-transfected cells, which was set arbitrarily at 1. Data are shown as the mean ± SD of at least three biological assays performed in triplicate.The statistical significance calculated by Student's t-tests is indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). whereas the transfectants over-expressing Notch1, Notch2 or Notch3 showed higher levels of glycerol release when compared with their respective control cells (Fig. 4B).No significant differences were observed in cells over-expressing Dlk2 or Notch4 (Fig. 4A,B).We also observed that 80-90% of 3T3-L1 adipocytes over-expressing Dlk1, Dlk2, Notch2, Notch3 or Notch4 genes contained larger lipid droplets than their corresponding controls or non-transfected 3T3-L1 differentiated cells (L1C) (Fig. 4C).However, differentiated Notch1 over-expressing cells, which show an increase in the number and size of adipocytes compared with their controls, exhibited a reduction in the multilocular lipid droplet size (Fig. 4C). To further characterize at a functional level the different types of adipocytes derived from each transfectant, we also analysed the lactate released into the extracellular medium, which indirectly provides the extra-cellular acidification rate (ECAR) (Fig. 4).Compared to their corresponding controls, differentiated 3T3-L1 transfectants over-expressing Notch1 or Notch4 showed lower levels of lactate released to the extracellular medium, whereas the differentiated transfectants over-expressing Notch3 released higher levels of lactate (Fig. 4F).No significant differences were observed in cells over-expressing Notch2, Dlk1 or Dlk2, or in non-transfected 3T3-L1 differentiated cells (Fig. 4D,E,F). Finally, we measured the rate of oxygen consumption (OCR), which is a measure of cellular respiration and mitochondrial function, by using a phosphorescent oxygen probe (Fig. 5).As shown, differentiation of 3T3-L1 cells into adipocytes increased the oxygen consumption rate (OCR) (Fig. 5A).Besides, we observed higher levels of OCR in Dlk1, Dlk2, or Notch1 transfectants when compared with their respective controls (Fig. 5B,C).On the other hand, the OCR change in 3T3-L1 transfectants over-expressing Notch2, Notch3 and Notch4 was not significant (Fig. 5D,E,F). ",
"section_name": "NOTCH receptors and DLK proteins modulate 3T3-L1 adipocyte browning. 3T3-L1 preadipo-",
"section_num": null
},
{
"section_content": "Figure 2) and inhibit NOTCH1 activation and signaling 36 , we considered important to study here whether DLK1 or DLK2 could block the observed proadipogenic effects of the over-expression of NOTCH1 in 3T3-L1 cells.For this purpose, we performed standard adipogenic assays with 3T3-L1 cells stably over-expressing the NOTCH1 receptor, cultured with control conditioned media or conditioned media containing sDLK1 or sDLK2 proteins during the entire adipogenic treatment.Soluble DLK proteins in the media were able to diminish the increment in the adipogenic potential of 3T3-L1 cells generated by the over-expression of Notch1, as indicated by a decrease in aP2 and Pparg expression (Supplementary Figure 3B).These results indicate that soluble DLK proteins can inhibit the 3T3-L1 adipogenesis process through the inhibition of NOTCH1 signaling. Following this observation, we decided to study whether DLK proteins could also affect the activation of the other three NOTCH receptors.We analyzed NOTCH-dependent transcriptional activity by luciferase assays in Balb/c14 cells transiently expressing the extracellular regions of DLK1 (DLK1E) or DLK2 (DLK2E), alone or in combination with one of the four full length NOTCH receptor genes (Fig. 6).The results indicated that transient over-expression of DLK1 or DLK2 extracellular regions inhibited NOTCH1, 3 and 4 signaling to different extents (Fig. 6A,C,D).DLK1 also seemed to strongly inhibit NOTCH2 signaling, whereas no significant differences were found in NOTCH2 signaling when DLK2 was transiently over-expressed (Fig. 6B).We could observe that the grade of inhibition caused by DAPT, a gamma-secretase complex inhibitor, was similar to that observed when each DLK protein was over-expressed (Fig. 6E).These results indicate that DLK proteins are able to inhibit the activity of the four mammalian NOTCH receptors to different extents, and suggest that the effects of NOTCH receptors on adipogenesis and adipocyte browning can be modulated by membrane and soluble DLK proteins. ",
"section_name": "The proteins DLK1 and DLK2 inhibit the activation and signaling of each one of the four mammalian NOTCH receptors. Since both DLK proteins inhibit adipogenesis of 3T3-L1 cells (Supplementary",
"section_num": null
},
{
"section_content": "The inhibitory effect of DLK proteins on all NOTCH receptors' activity, and additional published data confirming the inhibition of Dlk1 expression by NOTCH signaling 4,5 , suggested to us the existence of a complex feedback mechanism that would modulate the overall expression levels of DLK proteins and NOTCH receptors, as well as NOTCH activation and signaling.This feedback modulation could influence the adipogenic potential and the final adipocyte phenotype of 3T3-L1 cells.To analyze this potential feedback mechanism, we used our 3T3-L1 transfectant pools over-expressing each one of the four NOTCH receptors, the NOTCH target gene Hes1 or each one of the two DLK proteins (Figs 7 and8). We first observed that the over-expression of any of the four NOTCH receptors resulted in an increase in NOTCH-dependent transcriptional activity (Fig. 7A), which indicated that all NOTCH receptors were transcriptionally active in our cells.As expected, stable over-expression of NOTCH1, NOTCH3 and NOTCH4 in 3T3-L1 cells increased the expression of the NOTCH signaling target genes Hes1 and Hey1.However, NOTCH2 over-expression increased Hey1, but surprisingly inhibited Hes1 expression (Fig. 7B).This fact suggested to us that changes in Notch2 expression levels might influence the expression of the other three Notch genes, which, in turn, may affect the global NOTCH signaling and, ultimately, Hes1 expression.We show in Fig. 7C that over-expression of Notch1 exerted no significant effect on the expression of Notch3 and Notch4, but caused a small but significant decrease in Notch2 expression.Notch2 over-expression caused no significant changes in Notch4 mRNA levels, but dramatically increased the expression of Notch3 and decreased that of Notch1.On the other hand, over-expression of Notch3 up-regulated the expression of Notch1 and Notch2, but did not cause significant changes in Notch4 expression.Finally, Notch4 over-expression increased the expression of Notch3, but down-regulated that of Notch1 and Notch2.Thus, a variation in the expression of only one of the Notch genes seems to modulate the expression of the others in a way that could allow cells to reach a particular stoichiometry in the levels of global NOTCH activity. We also studied here whether the alteration of Notch expression could affect the expression of Dlk1 and Dlk2.We observed that over-expression of each Notch gene decreased Dlk2 expression in different amounts (Fig. 8A).Notch2 and Notch4 also decreased Dlk1 expression, but no significant changes were observed in Dlk1 expression levels following Notch3 over-expression.Surprisingly, Notch1 over-expression in these cells was accompanied by a significant increase in Dlk1 levels (Fig. 6A).NOTCH target genes, such as Hes1, whose expression has previously been inversely correlated with Dlk1 gene expression levels 5 , could mediate some of the observed effects of every NOTCH receptor signaling on Dlk1 and Dlk2 gene expression.Thus, we have observed that Hes1 over-expression in 3T3-L1 cells strongly decreased the expression of Dlk1, as published previously, and, to a lower extent, that of Dlk2 (Fig. 8B). We show here that the over-expression of Dlk1 led to a significant decrease in Hes1 and Hey1 expression.However, despite DLK2 also inhibiting NOTCH1, 3 and 4 receptors' transcriptional activity, no significant changes in Hes1 expression and even a slight increase in Hey1 expression were observed when Dlk2 was over-expressed in these cells (Fig. 8C).The slight effects of Dlk2 over-expression on Hes1 and Hey1 expression suggested to us that the response of 3T3-L1 cells to the inhibition of NOTCH signaling by DLK proteins could lead also to a change in the expression pattern of Notch1-4.Indeed, Dlk1 and Dlk2 over-expression led to variations in each Notch gene expression to different extents (Fig. 8D).Finally, we observed here that the over-expression of one Dlk gene affects the expression of the other (Fig. 8E), as described before 21,36 . All these data suggest that the expression of Notch and Dlk genes seems to be coordinated in the cells by a mechanism that globally regulates the NOTCH transcriptional activity of each NOTCH receptor and signaling to permit or not a particular biological process, such as white or brown adipogenesis, to proceed. ",
"section_name": "Feedback modulation of gene expression among NOTCH receptors and DLK proteins.",
"section_num": null
},
{
"section_content": "The role of NOTCH signaling in adipogenesis is still unclear.Different authors have claimed either null, positive, or negative effects on the adipogenesis of different cell lines, among them 3T3-L1 preadipocytes.Thus, some works suggested that the NOTCH signaling pathway is dispensable for adipocyte specification and differentiation from either mesenchymal or epithelial progenitors 1 .However, Garcés and coworkers showed that Notch1 expression and function were necessary for adipogenesis of 3T3-L1 cells 2 .A more recent work described that the intracellular active region of NOTCH4 also enhanced 3T3-L1 cell proliferation and adipogenesis, and decreased Dlk1 expression 4 .On the contrary, blocking NOTCH signalling with DAPT, an inhibitor of the gamma-secretase complex, enhances adipogenesis of differentiated mASCs at an early stage 46 .This effect may be due to depression of Dlk1/Pref-1 and promotion of PPAR-gamma activation, which works through the inhibition of NOTCH2-HES1 pathway by DAPT.Other authors demonstrated that the expression of the gamma-secretase complex, which activates NOTCH signaling, decreases during adipocyte differentiation, and ectopic expression of bovine PSENEN protein reduced the adipogenesis process in 3T3-L1 cells 47 .Urs and collaborators proposed an initial requirement of NOTCH signaling inactivation for preadipocyte cell commitment 6 .The adipocyte differentiation of 3T3-L1 cells was significantly reduced when cells constitutively expressed JAGGED1 or HES1 5 .Finally, recent work indicate that increased NOTCH signaling in mouse adipocytes results in the blockage of the expansion of white adipose tissue 48 .We hypothesize that all these contradictory results could be attributed to the need of the cells to reach a precise level of each NOTCH receptor activation leading to a stoichiometry of global NOTCH signaling permissive or not for adipogenesis. In this work, we studied the role of the NOTCH receptors and their non-canonical ligands, DLK1 and DLK2, in 3T3-L1 cells adipogenesis and adipocyte browning.We selected 3T3-L1 cells for these studies because they are one of the main cellular models of adipogenesis in which other authors have observed a high variability in the effects of NOTCH receptors and ligands.Our results show that stable over-expression of any of the four NOTCH receptors in 3T3-L1 preadipocytes enhances the adipogenic response of these cells.The lipolysis assays performed indicated that over-expression of the four NOTCH proteins increases the release of glycerol into the culture medium in response to isoproterenol, compared with their respective controls.Moreover, adipocytes showed different phenotypes depending on the Notch gene over-expressed.Thus, the over-expression of Notch2, Notch3 or Notch4 generated adipocytes with larger lipid droplets than controls, but Notch1-overexpressing adipocytes contained smaler multilocular lipid droplets as compared with its control cells.Thus, Notch1 overexpression, but not Notch 2-4 overexpression, seems to induce a brown-like adipocyte phenotype in 3T3-L1 cells. An increasing number of works by us and others have confirmed that DLK1 causes the inhibition of NOTCH signaling in different cellular processes 7,35,[38][39][40][41][42][43] .In previous works, we have demonstrated that DLK1 and DLK2 can inhibit the adipogenic process of 3T3-L1 cells 17,49 , and that one of the molecular partners that could mediate the function of DLK proteins in adipogenesis was the NOTCH1 receptor, whose activation and signaling was inhibited by both DLK proteins in a dose-dependent manner 21,36,39,43 .We hypothesized here that DLK proteins could inhibit not only the activation and signaling of NOTCH1, but that of the four mammalian NOTCH receptors to different extents, thus generating a global level of NOTCH signaling permissive or not of the adipogenesis process.In agreement with this hypothesis, in this work we have demonstrated that soluble DLK1 and DLK2 proteins, as it is also the case with the membrane variants, reduce the augmented adipogenic potential of 3T3-L1 cells that over-express the NOTCH1 receptor.These results are in agreement with recent work from other authors demonstrating that blockage of the NOTCH1 receptor by DLK1 modulates the size of adipocytes in vivo 7 .As expected, we found that the over-expression of Dlk1 in these cells decreases the amount of glycerol released from adipocytes into the culture medium in response to isoproterenol, which indicates that these adipocytes foster a lower lipolytic activity despite showing larger lipid droplets than control cells.Alternately, these adipocytes may have accumulated lower lipid levels throughout their adipogenic process.However, Dlk2-over expressing adipocytes also show large lipid droplets, although the release levels of glycerol were not significantly different.Moreover, we have also demonstrated that the DLK1 and DLK2 proteins inhibit the activity of each one of the four mammalian NOTCH receptors to different degrees, with the exception of NOTCH2, which was only inhibited by DLK1 but not by DLK2. Even though 3T3-L1 preadipocytes have always been considered a well-known cellular model of white adipogenesis, Morrison and McGee observed that 3T3-L1 adipocytes display phenotypic characteristics of multiple adipocyte lineages, express Pgc1a and increase oxygen consumption and the expression of brown adipocyte genes in response to catecholamines 44 .In agreement with these authors, we have observed that the expression of several brown adipogenic markers is increased in 3T3-L1 adipocytes and the size and distribution of their lipid droplets is more coherent with that of brown or beige adipocytes.Despite a decreased expression of Cidea and Sirt1, differentiated 3T3-L1 cells show increased levels of Ucp1, Pgc1a, Gyk and Prdm16 markers.These results, together with the multilocular small lipid droplets observed in these cells, are more consistent with a brown-like phenotype, as other authors have described 50 .The Pparg transcription factor is considered both as an adipocyte marker in 3T3-L1 cells and as a factor that favors adipocyte browning by activating Ucp expression 51 .Therefore, the induction of Pparg expression after 3T3-L1 differentiation also supports the idea that 3T3-L1 adipocytes show a brown-like phenotype.As expected, the level of glycerol release and the respiration rate were generally higher in differentiated adipocytes than in undifferentiated 3T3-L1 cells; however, not significant differences were observed for lactate output in 3T3-L1 adipocytes 44,52 . The brown adipose tissue (BAT) functions by generating heat through mitochondrial uncoupling proteins, in particular UCP1 and UCP3 53 .The role of NOTCH signaling on brown adipogenesis is also controversial.Thus, some authors showed that inhibition of NOTCH signaling promotes browning and alleviates obesity 8,9 .On the contrary, Pasut and co-workers demonstrated that, subsequent to the deletion of the transcription factor PAX7 (Paired Box 7) and following acute muscle injury, NOTCH signaling promoted the differentiation of satellite cells into brown adipocytes rather than into a skeletal muscle cell phenotype 10 .Some works have also shown a role for DLK1 in adipocyte browning and suggest a possible involvement of DLK2 in this process.Thus, the expression of Dlk1 is high in fetal BAT and declines after birth, although Dlk1-null mice showed unaltered BAT fetal development, with an over-activation of thermogenesis in the postnatal period.As it occurs in 3T3-L1 preadipocytes, Dlk1 expression decreases after the induction of the differentiation in brown preadipocyte cell lines 33 .Other authors described that Dlk1 inhibits thermogenesis in brown adipocyte models 34 .Young women with cold-activated brown adipose tissue have lower serum DLK1 than women lacking brown adipose tissue 54 . It is believed that an increase in the expression of brown-fat signature genes and in the number of mitochondria or in mitochondrial biogenesis and function are key features of BAT.Interestingly, we have observed that Notch1 over-expression in differentiated 3T3-L1 cells upregulates the expression of Ucp1, Pgc1a, Gyk, Prdm16, Cidea, Pparg and Sirt1, and there is more amplification of CytB than in control cells, whereas their expression or amplification is down-regulated or it does not change when Notch2 or Notch3 are over-expressed in these cells.In the case of Notch4 transfectants, the expression of Ucp1, Pgc1a, Gyk, and Pparg markers is down-regulated in differentiated cells, and the amplification of CytB is also lower than control.However, unexpectedely, the expression of Prdm16, Cidea and Sirt1 is increased in Notch4 transfectants.CIDEA is a multifunctional protein, highly expressed in brown adipose tisusue, with a clearly defined role in the promotion of lipid droplets in brown and white adipocytes 50 .These data suggest that Notch1 may promote differentiation into 3T3-L1 brown adipocytes, whereas Notch2, Notch3 and Notch4 may promote 3T3-L1 cells to differentiate toward the white phenotype.It has been described that brown adipocytes showed higher levels of OCR and ECAR when compared with control cells 55 .We observed that only Notch1 adipocytes showed significantly higher levels of OCR and diminished levels of lactate released into the culture medium.Our findings may imply that lactate could be transported to mitochondria and used as a fuel substrate.Interestingly, a recent study described that lactate was a major substrate for the TCA cycle in several tissues, including adipocytes and glucose feeds the TCA cycle via circulating lactate 56 .Besides,the increased number of mitochondria in Notch1-over-expressing cells would lead to a greater use of lactate as fuel in these cells.This suggests that only Notch1-overexpressing adipocytes could undergo functional lineage transformation toward a brown adipocyte phenotype.All these data suggest that Notch1-over-expressing transfectants could have developed smaller lipid droplets than their empty vector-transfected control adipocytes because of a higher expression of UCP1 and increased mitochondrial biogenesis, which would lead to a higher OCR and lipolysis rate. On the other hand, we have observed that differentiated 3T3-L1 cells over-expressing Dlk1 show decreased levels of Cidea and Sirt1 and increased expression levels of Pgc1a and Gyk, although no significant changes in Ucp1 and Prdm16 expression were observed.The expression of Gyk, Cidea and Sirt1 decreases in stable Dlk2-over-expressing transfectants, but no significant changes were observed in Pgc1a, Ucp1 or Prdm16 expression.Therefore, these transfected cells, when differentiated, do not show evidence of a gene expression profile typical of brown adipose cells, despite the increase in Pgc1a and Gyk expression in Dlk1-stably transfected cells.Besides, the levels of mitochondrial CytB in Dlk2 transfectants, but not in Dlk1 transfectants, are lower than in control cells, although, as it occurs with Notch1 transfectants, both Dlk1 and Dlk2 transfectants show higher OCR than control cells.It has been described that DLK1-overexpressing mice showed increased oxygen consumption, indicating an increase in energy expenditure 29 .These slight and contradictory effects could be probably explained by a differential inhibition of each NOTCH receptor´activity, since each NOTCH receptor exerts different effects on the expression of these markers.We think that qualitative and quantitative differences in overall NOTCH signaling may place the cells in different states allowing them to interpret the same extracellular signals so that they differentiate or not to at least two different adipocyte phenotypes. Finally, in this work we have also revealed the existence of a complex feedback mechanism that modulates the overall expression levels of the Notch and Dlk genes and the global NOTCH activation and signaling.Each NOTCH receptor increases the level of global NOTCH signaling and induces the expression of the NOTCH signaling targets Hes1 and Hey1, except for Notch2, which unexpectedly reduces Hes1 mRNA levels.Furthermore, stable over-expression of each one of the NOTCH receptors in 3T3-L1 cells influences the expression levels of the other Notch genes to different extents.This interplay among Notch members, specifically between Notch1 and Notch3, has been shown by other authors 57,58 .We have also shown here that DLK proteins inhibit each one of the four NOTCH receptors' activity to different extents and that the over-expression of Dlk1 inhibits the global NOTCH signaling by reducing the level of expression of Hes1 and Hey1.The observed unexpected effects of stable Dlk2 over-expression on Hes1 and Hey1 suggest that the potential inhibitory effect of the stable over-expression of Dlk2 on NOTCH signaling could be achieved through other factors different from HES1 and HEY1. NOTCH signaling can both positively and negatively regulate canonical ligand expression, such that defects in NOTCH signaling are associated with increased expression of DLL1 59 or DLL4 60 .As other authors have indicated 5 , non-canonical ligands such as DLK1 are also regulated by NOTCH signaling.In this work we have observed that NOTCH receptors' activity modulate Dlk1 and Dlk2 expression levels to different extents.Besides, the results presented here demonstrate that Hes1 over-expression in 3T3-L1 cells inhibits the expression of both Dlk2 and Dlk1.These data are in agreement with those of Ross and coworkers, who demonstrated that HES1 seemed to down-regulate Dlk1 expression, which would permit adipogenesis of 3T3-L1 cells to complete 5 . Importantly, it has been reported that the expression of Dlk1 and Dlk2 is coordinated in 3T3-L1 cells and that DLK1 and DLK2 proteins interact and may inhibit each other's activities 21,36,39,43 .Indeed, in this work, we have observed that the over-expression of one Dlk gene affects to the expression of the other.Therefore, it is also possible that, depending upon the different expression levels of DLK1 and DLK2 in these cells, both proteins may lead either to a decrease or to an increase in the global NOTCH signaling levels, depending on their stoichiometry and their interaction affinities.This kind of coordination of the expression and competition between NOTCH ligands has been also described by other authors 61 .Furthermore, we have observed that the over-expression of Dlk1 and Dlk2 increases the expression of endogenous Notch genes to different extents, suggesting a response of the cells aimed at re-equilibrating global NOTCH signaling.Our data reveal that the interplay among the NOTCH receptors and DLK proteins in 3T3-L1 cells affect the expression of each one of these genes, and suggest that the expression of the NOTCH canonical and non-canonical ligands participates in these complex feed-back mechanisms.Further analysis, beyond the scope of this work, will be needed to study in detail the coordination in the expression of these genes in 3T3-L1 preadipocytes. In summary, the data presented here deepen into the understanding of the role of NOTCH and DLK proteins on the control of 3T3-L1 adipogenesis and adipocyte fate.In Fig. 9, we schematically summarize the potential effects of NOTCH receptors and DLK proteins on 3T3-L1 adipogenesis and preadipocyte whitening/browning.We believe that continuing with the study of the molecular pathways involved in this cell differentiation process and the coordination among the four NOTCH signaling activities and their modulation by canonical and non-canonical ligands would permit to advance in the development of novel and promising anti-obesity therapies. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Plasmids.Bacterial cultures, plasmid DNA isolation and purification, and bacterial transformation and amplification of TOP10 Escherichia coli competent cells was performed as previously described 36 .Plasmids pCDLK1 (DLK1) and pCDLK2 (DLK2) derivate from pCD2 vector (V1) and contain the complete cDNA sequence of either Dlk1 or Dlk2 in sense orientation, respectively 17 .Plasmid pC-N1 (N1) derivates from pCD2 (V1) and contains the complete mouse Notch1 cDNA (ATCC clone: MBA-105) in sense orientation 36 .Plasmid pCN-N2 (N2) (A gift from Dr. Anna Bigas, IMIM, Barcelona, Spain) contains the complete mouse Notch2 cDNA cloned into the EcoRI-NotI restriction sites of the pCDNA3 expression vector (V2).Plasmid pEntry-N3 (N3) contains the complete mouse Notch3 cDNA sequence in pCMV6-Entry vector (Origene) (V3).pG-N4 (N4) contains the complete mouse Notch4 cDNA sequence cloned into the HindIII-XbaI restriction sites of the pEGFP-N1 vector (Clontech) (V4).Plasmid pN-HES1 (H1) expresses the complete HES1 protein 36 .Plasmids pL-DLK1 (DLK1E) and pL-DLK2 (DLK2E) contain de cDNA of the extracellular regions of DLK1 and DLK2 36 . All plasmids were confirmed by restriction analysis and sequenced by Macrogen.The oligonucleotides used for sequencing were obtained from Bonsai Technologies. Cell culture and cell transfections.Mammalian cell lines were cultured as describe before 17,36 .The cell lines used were: 3T3-L1 (L1; ATCC CCL-92.1);HEK 293 T/17 (ATCC CRL-11268); and Balb/c14 (Balb/c 3T3 sub-clones negative for Dlk1 expression 62 .Stable and transient transfections were performed as previously described 36 . Quantitative RT-PCR expression analysis.Confluent cell monolayers were processed as previously described to obtain total RNA and cDNAs 36 .Gene expression assays by qRT-PCR were performed as previously described 36 .P0 expression was used as the control to compare the C T from the different samples in all qPCR experiments 63 .The qRT-PCR primers to determine Dlk1, Dlk2 and Hes1 expression levels were previously described 17,21 .The expression of Notch genes and Hey1 gene was analyzed with the oligonucleotides showed in Table 1 (Bonsai Technologies). Conditioned media.HEK 293T/17 cells were transiently transfected with plasmids pL-DLK1 (DLK1E) and pL-DLK2 (DLK2E), which express the soluble forms of DLK1 (sDLK1) and DLK2 (sDLK2), respectively.Protein expression and secretion of these proteins were analyzed as previously described 36 (Supplementary Figure 3A). Protein sample preparation and Western blotting.Protein samples were obtained, quantified and electrophoresed as previously described 36 .Western blot was performed by using an appropriated dilution of the primary and the secondary antibodies (usually, 1: 2,000) (Table 2).Detection of alpha-tubulin with a specific antibody (Sigma) was used as a protein loading control. Luciferase assays.NOTCH transcriptional activity was analyzed by luciferase assays in Balb/c14 cells and stably transfected 3T3-L1 pools as previously described 36 .When cells were transiently co-transfected with DLK expression plasmids, the assays were performed in Balb/c14 cells because these cells do not express DLK1 and express very low levels of DLK2, and have higher transient transfection efficiency compared to 3T3-L1 cells.To analyze the effect of DLK proteins on NOTCH activity, we performed these assays by co-transfecting Balb/c14 cells with a Notch expression plasmid and with plasmids expressing sDLK1 or sDLK2 proteins.We also treated Balb/c14 cells with the gamma-secretase inhibitor DAPT (10 μM) as a NOTCH signaling inhibition control.Transfected cells were processed as previously described 36 . Adipogenic assays.The induction of 3T3-L1 adipogenesis and the staining of adipocytes with red oil O was performed according to standard procedures as previously described 17 .Sometimes, cells were induced to differentiate in the presence of control conditioned media or conditioned media containing sDLK1 or sDLK2 during the entire differentiation process.Assays were repeated at least three times.We determined the level of differentiation by analyzing the expression of the late adipocyte differentiation marker aP2 and the intermediate marker Pparg, as previously described 64 , seven days after induction of adipogenesis.To study the phenotype of 3T3-L1 adipocytes, we also analyzed in the same samples the expression of the following brown adipose markers: Ucp1 (coding for uncoupling protein-1); Pgc1a (Peroxisome proliferator-activated receptor-gamma coactivator); Gyk, encoding for a glycerol kinase activated in brown adipocytes and involved in triglyceride and glycerophospholipid synthesis 65 ; Prdm16, thought to function as a key transcriptional co-regulator of brown cell adipogenesis function 55 ; Cidea, highly expressed in lipid droplet membranes and mitocondria of brown adipocytes,involved in the browning process and considered as a BAT differentiation marker 45 ; and Sirt1, a mitochondrial biogenesis marker 66 .Pre-designed qRT-PCR oligonucleotides of these genes were obtained from Sigma (Table 3). We also analyzed the biogenesis of mitochondria by qPCR analysis of the ratio of the levels of the mitochondrial gene CytB and the genomic gene ApoB in terminal differentiated cells by using oligonucleotides previously described 67,68 . The amount of glycerol released into the culture medium was determined using the lipolysis colorimetric assay kit (BioVision).The extracellular lactate was measured using the lactatate colorimetric assay kit (BioVision).Finally, to measure the oxygen consumtion rate (OCR), we used the oxigen consumption rate assay kit (AbCam).In these three last assays, we seeded 15,000 cells per well in 96-well plates, then we proceeded with the adipogenic differentiation protocol and, finally, we performed the assays following the manufacturer's recommendations.Data were normalized with total protein amount or the number of cells seeded in each well. Equipment and Settings.RNA and DNA concentration and purity (20 nm/280 nm) were analyzed by using the spectrophotometer NanoDrop One (Thermo Scientific). Quantitative RT-qPCRS were performed with StepOne Plus RT-qPCR system (Applied Biosystems) by using Fast SYBR green mix.The results were obtained by using the StepOne software 2.3 with the parameters recommended by the company, analyzing always the melting curve for each gene. Microscopy images were visualized with an objective 40X of Motic AE31 microscopy connected to a Moticam 2300 camera (3.0 M Pixel USB 2.0).Cell images, 400X magnification, were acquired with the software Motic Images Plus 2.0 with the standard parameters (Exposition: 418.9, Gamma: 0.8019). Western blot images s were obtained by developing exposed films (CP-BU New, Agfa) for 30 seconds (Tubulin), 1 minute (NOTCH1, NICD1, NOTCH2), 5 minutes (NOTCH3) or 10 minutes (NOTCH4), with the Pierce ECL Plus Western Blotting substrate kit (Thermo Scientific) in a Curix 60 developing apparatus (AGFA).Films were scanned with HP Officejet Pro 8600 scanner and signals of the different proteins were quantified by using the QuantityOne 4. 6. 5. (Basic) software.We analyzed the intensity of the signal per mm 2 with a Volume Rect Tool.Blots for the Supplementary Figure 1 were converted to a grey color. Colorimetric determinations to quantify total protein amount, extracellular lactate in the culture medium, and the release of glycerol to the the medium were performed with a plate reader Axis UVM340 (Biochrom).Table 2.Primary and secondary antibodies used in Western blot analysis. To measure the OCR of the different cultured adipocytes, we used a fluorescence spectrophotometer F-7000 (Hitachi).Luciferase assays were measured by using a Monolight 3096 Microplate Luminometer (Becton Dickinson) and samples were processed with the Dual-224 Luciferase Reporter Assay System (Promega), following the supplier's recommendations. ",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Data are presented as the mean ± S.D of at least three different independent assays performed in triplicate.Data were also analyzed with Student's t test to determine statistical significance.A P value of ≤0.05 was considered statistically significant (*); a P value ≤0.01 was considered highly statistically significant (**); and a P value of ≤0.001 was considered extremely statistically significant (***). ",
"section_name": "Statistical analysis.",
"section_num": null
}
] |
[
{
"section_content": "We thank Dr. Anna Bigas from IMIM (Barcelona, Spain) for her gift of the complete NOTCH2 expression plasmid.We thank Dr. Marta Casado, from Instituto de Biomedicina de Valencia (Valencia, Spain), for the information about the oligonucleotides used to amplify the genomic gene ApoB and the mitochondrial gene CytB, and the protocols to obtain total DNA.We also thank the laboratory technician Ms. María-Ángeles Ballesteros for her invaluable help.This work was supported by funds from the Ministry of Economy and Competitiveness of Spain (BFU2010-16433), and by funds PII1I09-0164-00 and PEII11-0062-2456 from the Health Council of the Regional Government of Castilla-La Mancha (Spain), supported by the Fondo Europeo de Desarrollo Regional (FEDER). ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "V. Baladrón Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-35252-3. 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": "Author Contributions",
"section_num": null
},
{
"section_content": "V. Baladrón ",
"section_name": "Author Contributions",
"section_num": null
},
{
"section_content": "Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-35252-3. ",
"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
}
] |
10.1186/1742-4690-9-s1-p24
|
CD4.CD8 ratio decrease in AIDS, explained by a molecular mimicry between African HIV-1 Nef and Notch-1. Nef as a target for vaccine and NF-Kb inhibitors (salicylate, resveratrol,curcumin, epigallocatechine-3-gallate)
|
Background The AIDS hallmark is the simultaneous fall in CD4 and rise in CD8 T lymphocytes. Interestingly, this very pathognomonic but unexplained decrease of CD4/CD8 ratio is also characteristic of a member of the EGF family, Notch1 function (Fowlkes BJ, 2002). Calenda V (1994) found that Nef hampered drastically bone marrow progenitors cells functionality. African HIV-1 strain NDK (Spire B, 1989), which induced a fulminant AIDS killing the patient in only 15 days, decreases dramatically CD4 counts. Nef is the most abundant HIV-1 protein in infected cells (85% of mRNA). Nef is a superantigen, its action is amplified 10,000 times compared to a common antigen.
|
[
{
"section_content": "The AIDS hallmark is the simultaneous fall in CD4 and rise in CD8 T lymphocytes.Interestingly, this very pathognomonic but unexplained decrease of CD4/CD8 ratio is also characteristic of a member of the EGF family, Notch-1 function (Fowlkes BJ, 2002).Calenda V (1994) found that Nef hampered drastically bone marrow progenitors cells functionality.African HIV-1 strain NDK (Spire B, 1989), which induced a fulminant AIDS killing the patient in only 15 days, decreases dramatically CD4 counts.Nef is the most abundant HIV-1 protein in infected cells (85% of mRNA).Nef is a superantigen, its action is amplified 10,000 times compared to a common antigen. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "We found previously Notch-1 in the LTR (Long Terminal Repeat) of another retrovirus [Mouse Mammary Tumor Virus (MMTV)] (Tran MKG, Eurocancer, Paris, 1999).As Nef is located also in HIV-1 LTR, we looked for Notch-1 in Nef. ",
"section_name": "Objective",
"section_num": null
},
{
"section_content": "Amino Acid (AA) alignment between Epidermal Growth Factor (EGF) family members (including Notch-1) and Nef (Los Alamos HIV sequences Database, 2002). ",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Nef COOH-terminus of HIV-1 clade D African strains (from Congo Democratic Republic, Chad, Tanzania, Uganda, South Africa, Kenya,…), but not from other parts of the world (other non-D clades), was a perfect molecular mimetic of Notch-1: They shared a heptapeptide (7 AA) SRLAFEH.The homology between Nef (Poon AFY, 2009) and Notch (BLASTP on mouse Notch-1) chimera was 67 AA long with 4 His, 1 Cys and 1 Trp (highly significant): Nef : GWCFEVEEDTE-GETNSLLHPISQHGMEDPERQVLVWRFNSRLAFEH-KARLMHPEFYKNC Notch : GWLLD…FEQDSEG ETNSLPHLISQHAL ANPEMQALA-HGKSRLAFEH QVRLSHLPVANNC It included the Nef LL and ED doublets precisely implicated in CD4 down-regulation and EE in β-COP recruitement (Benichou S.1994). ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "This opens new avenues for a vaccine targeted to Nef-Notch specific to Africa, a continent devastated by AIDS and tuberculosis (in South Africa, about 60% HIV-1 infected patients had also tuberculosis). ",
"section_name": "Conclusions",
"section_num": null
}
] |
[] |
10.5937/opmed2102040d
|
Chronic lymphocytic leukemia in general practice
|
<jats:p>Introduction: Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults, rarely affecting children. It is more common in males over 60. Etiopathogenetically, it represents an abnormal proliferation of lymphocytes in the bone marrow, which are dysfunctional although morphologically similar to mature ones. Case report: Female patient, 66, a housewife, comes for an examination due to fatigue lasting about a month and weight loss of 3-4 kg. She denies other health problems. On the examination, she is alert, oriented to time, space, and persons, eupnoeic, lymphadenopathy on the neck, axillae and groins. She gives away the impression of a patient with mild clinical symptoms. Clinical examination: clear breath sounds in all lung fields, regular heartbeat, clear tones, BP 140/80 mmHg, abdomen at chest level, painless to palpation, liver not palpable, and spleen palpated 2 cm below the costal arch. CBC (complete blood count) showed an increased number of leukocytes 181.30x109 /L and lymphocytosis 92.2%, other parameters were normal. She was referred to the hematologist in the Kraljevo General Hospital, where further diagnostics were performed. Blood count was repeated, Chest X-ray was performed, ultrasound (US) of the abdomen neck, axillae, groins, and heart, virology tests, ENT examination. Abdominal US showed a pathological finding with enlarged liver, spleen, lymph nodes (LN). ENT examination: enlarged tonsils, other findings unremarkable. Since chronic lymphocytic leukemia was suspected, she was referred to the Clinical Center of Serbia. Immunophenotyping (IF) and computed tomography (CT) of the neck, chest, and abdomen were performed. The diagnosis of CLL was confirmed. A Medical Council decided to perform immunochemotherapy (IHT) with fludarabine and rituximab. After 6 received cycles, the patient feels well and the CBC parameters are normal. Conclusion: CLL may be asymptomatic or nonspecific and with mild symptoms in a large number of patients. Therefore, blood tests with leukocytosis and absolute lymphocytosis findings are crucial to suspect the disease and perform further diagnostics.</jats:p>
|
[
{
"section_content": "Leukemije su maligne bolesti krvi.Prema dužini trajanja dele se na akutne i hronične, a prema vrsti leukocita koji maligno bujaju, na granulocitne i limfocitne.Predstavljaju nenormalno, generalizovano razmnožavanje jedne od leukocitnih loza, često praćeno povećanim brojem leukocita u perifernoj krvi 1 . Hronična limfocitna leukemija (HLL) je maligno oboljenje krvnih ćelija -limfocita.Morfološki se malo razlikuju od normalnih, ali su nefunkcionalni, što se posledično odražava na imunitet.Postoje dve vrste HLL: B ćelijska, koja je češća i javlja se kod 95% pacijenata i T ćelijska koja je ređa 1 .Etiologija je nepoznata.Okrivljuje se jonizujuće zračenje, genetika, patogeni agensi, virusi, bakterije, pesticidi i herbicidi, stres.Postoje dokazi da je učestalo izlaganje pesticidima i herbicidima glavni spoljašnji faktor rizika 1,2 .Porodična istorija bolesti može povećati rizik do tri puta više nego kod onih koji nemaju pozitivnu porodičnu anamnezu.Hronična limfocitna leukemija je, uglavnom, bolest muškaraca starijeg životnog doba, preko šezdeset godina.Rizik od HLL se značajno povećava sa godinama života.Postoji oko 13 novih slučajeva na 100.000 stanovnika starosti preko 65 godina 1,2 . U kliničkoj slici se javljaju uvećane limfne žlezde, uvećana jetra i slezina, ali i mnogi atipični znaci kao sto su umor, noćno znojenje, gubitak težine, osećaj nadutosti, svrab i hiperpigmentacija kože, herpes zoster i drugo.Određivanje stadijuma može se izvršiti po Rai i Binet klasifikaciji.Postoje ćetiri stadijuma: nulti kada je prisutna samo limfocitoza, I stadijum kada pored limfocitoze postoji i limfadenopatija, II stadijum limfocitoza, limfadenopatija i uvećana jetra i/ili slezina.U III stadijumu, pored navedenog, postoji i anemija a u IV stadijumu postoji još i trombocitopenija 1,2 .Početak bolesti je obično podmukao i HLL se često dijagnostikuje slučajno pri rutinskom pregledu krvne slike ili pri obradi asimptomatske limfadenopatije 2 .Pregledom krvne slike nađe se leukocitoza sa izraženom limfocitozom.U leukocitarnoj formuli procenat limfocita se u početku kreće od 60% do 75% a kasnije i do 90%.Iako zreli, ovi limfociti su atipični, nefunkcionalni sa posledicama na imunološki sistem.U većini slučajeva pacijent sa povećanim brojem leukocita se dobro oseća.U ređim slučajevima klinički simptomi i znaci prate početak bolesti. ",
"section_name": "Uvod",
"section_num": null
},
{
"section_content": "Leukemias are malignant blood diseases.In relation to the duration, they are divided into acute and chronic, and according to the type of leukocytes, which flourish malignantly, into granulocytic and lymphocytic.They represent an abnormal, generalized proliferation of one of the leukocyte lines, followed very often by an increased number of leukocytes in the peripheral blood 1 . Chronic lymphocytic leukemia (CLL) is a malignant disease of the blood cells-lymphocytes, which are morphologically slightly different from normal cells but are nonfunctional, which consequently affects immunity.There are two types of CLL: B-cell, which is more common and occurs in 95% of patients, and T-cell, which is less common 1 .The etiology is unknown.The culprits are ionizing radiation, genetics, pathogenic agents, viruses, bacteria, pesticides and herbicides, stress.There is evidence that frequent exposure to pesticides and herbicides is a major external risk factor 1,2 .A family history of the disease can increase the risk by up to 3 times than in people who do not have a positive family history.Chronic lymphocytic leukemia is a disease mainly of older men, over 60.The risk of CLL increases significantly with age.There are about 13 new cases per 100,000 people over the age of 65 1,2 . The clinical presentation is remarkable for enlarged lymph glands, liver, and spleen, but also many atypical signs such as fatigue, night sweats, weight loss, bloating, itching, and hyperpigmentation of the skin, herpes zoster, and others.Disease staging can be performed according to Rai and Binet classification.There are 4 disease stages: Zero, when only lymphocytosis is present, I when there is lymphadenopathy in addition to lymphocytosis, II lymphocytosis, lymphadenopathy, and enlarged liver and/or spleen.In stage III, in addition to the above, there is also anemia, and in stage IV there is also thrombocytopenia 1,2 .The onset of the disease is usually insidious and CLL is often diagnosed accidentally during a routine blood check or treatment of asymptomatic lymphadenopathy 2 .CBC reveals leukocytosis with pronounced lymphocytosis.Leukocyte formula initially shows lymphocyte percentages ranging from 60%-75%, and later up to 90%.Although mature, these lymphocytes are atypical, dysfunctional with consequences for the immune system.In most cases, the patient with an increased number of leukocytes feels well.In rare cases, clinical symptoms and signs accompany the onset of the disease.Normally, the bone marrow is made up of stem cells, which become mature blood cells over time.In CLL, too many blood stem cells become abnormal lymphocytes that do not reach a certain maturity and thus lose function.As the number of leukemic cells increases in the peripheral blood, there is a consequent increase in the number of cells in the bone marrow, thereby reducing the space for normal cells 1 ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "Pacijentkinja stara 66 godina, domaćica, dolazi na pregled zbog zamora koji traje oko mesec dana, u poslednja dva meseca izgubila je 3 kg do 4 kg, ostale tegobe negira.Leči se od povišenog pritiska, retko dolazi kod izabranog lekara, dugogodišnji je pušač, 20 cigareta dnevno, ne pije alkohol, u poslednjih nekoliko meseci imala je više stresnih situacija.Porodična anamneza pozitivna za kardiovaskularne bolesti i dijabetes. Bolesnica je svesna, orijentisana u vremenu, prostoru i prema ličnostima, afebrilna, eupnoična, pokretna, normalno uhranjena, koža bledo prebojena, prisutna limfadenopatija u vratu, aksilama i ingvinumu do 4 cm u konglomeratima, odaje utisak lakšeg bolesnika 1,2 . Objektivnim pregledom nađeno je: glava uobičajene konfiguracije, nos prohodan, jezik vlažan i neobložen, tonzile uvećane, očni bulbusi pokretni u svim pravcima, zenice reaguju na svetlost i akomodaciju, vrat pokretan u svim pravcima, štitasta žlezda u fiziološkim granicama, grudni koš simetričan, respiratorno obostrano pokretan, na plućima vezikularno disanje bez propratnih šušnjeva, srčana akcija ritmična, tonovi jasni bez patoloških šumova; TA 140/80 mmHg.Abdomen u ravni grudnog koša, palpatorno bolno neosetljiv, jetra se ne palpira, slezina se palpira za 2 cm, čvrsta, glatkih ivica, bezbolna.Bubrežne lože neosetljive na grubu perkusiju.Ekstremiteti bez edema i deformiteta, očuvane periferne arterijske pulsacije na GE i DE.Bez vidljivih neuroloških ispada. Urađena je kompletna laboratorija koja je pokazala visoke vrednosti leukocita 181,30x10 9 /L u perifernoj krvi, visoki procenat limfocita 92,2%.Ostali nalaz bio je u referentnim granicama.Odmah je upućena hematologu Opšte bolnice u Kraljevu, gde je hospitalizovana 15.10.2019.godine i gde je urađena dalja dijagnostika: radiološki pregled (RTG) pluća, ultrazvuk (EHO) abdomena, vrata, aksila, ingvinuma i srca, kompletna biohemija i ELIZA test (ELISA enzyme-linked immunosorbent assay); pregled ORL lekara, EHO abdomena: jetra je veličinom u granicama fizioloških vrednosti, u njoj se uočavaju dve nejasno definisane, hiperehogene promene promera 14 mm i 15 mm koje mogu biti hemangiomi, ali se ne isključuju ni lezije sekundarne etiologije.Slezina je veličinom na gornjoj granici fizioloških vrednosti (120 mm x 55 mm) izrazito heterogene strukture, infiltrativno je izmenjena sa vidljivim difuzno raspoređenim hipoehogenim mikroarealima koji su promera od 3 mm do 12 mm.U retroperitoneumu se zapaža veći broj izmenjenih limfnih nodusa (LN) promera 15 mm do 27 mm koji su hipoehogene strukture, ostali nalaz uredan, EHO vrata, aksilarne i ingvinalne regije: štitasta žlezda je veličinom u granicama normale, simetrična, regularnih kontura, homogene strukture, u desnom lobusu uočava se heterogeni cistično izmenjen nodus 7,5 mm x 4,5 mm a u levom lobusu izoehogeni nodus veličine 5 mm x 6 mm.Sub-lymph nodes, liver, and spleen as well as in other organs.The evolution of CLL is long, acutisations are rare and patients have problems with frequent infections, general deterioration due to the autoimmune phenomena, frequent hemolytic crises, or consequences due to bone marrow insufficiency. ",
"section_name": "Prikaz slučaja",
"section_num": null
},
{
"section_content": "A 66-year-old female patient, a housewife, comes for an examination due to fatigue that lasts for about a month.She lost 3-4 kg in the last two months, denies other health problems.She is treated for high blood pressure, rarely visits her GP, long-term smoker, 20 cigarettes a day, does not drink alcohol, had more stressful situations in recent months.She has got a family history of cardiovascular disease and diabetes. She is alert, oriented to time, space, and persons, afebrile, eupnoeic, mobile, well-fed, with pale skin, lymphadenopathy on the neck, axillae, and groins up to 4 cm, in conglomerates.She gives away the impression of a patient with mild clinical symptoms. Physical examination revealed: head of normal configuration, nose passable, tongue moist and uncoated, tonsils enlarged, eyeballs moving in all directions, pupils reacting to light and accommodation, neck movable in all directions, thyroid gland within physiological limits, chest symmetrical, respiratory bilaterally mobile, clear breath sounds in all lung fields, regular heartbeat, clear tones, without pathological noises, BP 140/80 mmHg.Abdomen in the plane of the chest, painless to palpation, the liver is not palpable, the spleen is palpated by 2 cm, firm, smooth edges, painless.Renal lodges are insensitive to rough percussion.Limbs without edema and deformity, preserved peripheral arterial pulsations on UE (upper extremities), and LE (lower extremities).No visible neurological deficits. A CBC showed high values of leukocytes 181.30x10 9 /L in peripheral blood with a high percentage of lymphocytes 92.2%.Other findings were within the reference range.She was immediately referred to a hematologist at the Kraljevo General Hospital, where she was hospitalized on October 15, 2019, and further diagnostics were performed: chest X-ray, ultrasound (US) of the abdomen, neck, axillae, groins, and heart, complete biochemistry and ELISA test (ELISA enzymelinked immunosorbent assay).ENT examination was performed.Abdomen US findings: the size of the liver is within the physiological limits; two vaguely defined, hyperechoic formations of 14 mm and 15 mm in diameter can be observed, which might be hemangiomas, but they also might be lesions of secondary etiology.The size of the spleen is at the upper limit of physiological values (120 mm x 55 mm) with a distinctly heterogeneous structure, it is infiltratively altered with visible diffusely distributed hypoechoic micro areas with a diameter of 3 mm to 12 mm.In the retroperitoneum, a larger number of altered lymph nodes (LN) with a diameter mandibularno se uočava par uvećaih LN, od kojih su neki izmenjene strukture, zbrisanog hilusa, promera 10 mm i 7 mm.Obostrano parajugularno uočava se više pojedinačnih LN u nizu, izmenjene strukture, zbrisanih hilusa, očuvanog L/W indeksa, najveći promer do 14 mm.Obostrano aksilarno uočava se više grupisanih uvećanih LN, ali očuvane strukture i hiperehogenog hilusa, očuvanog L/W indeksa, promera do 20 mm.Obostrano ingvinalno uočava se više grupisanih uvećanih LN, očuvane strukture i hiperehogenog hilusa, očuvanog L/W indeksa promera do 23 mm.Nalaz Kompjuterizovana tomografija-CT pregled vrata, grudnog koša i abdomena: u regiji vrata na svim nivoima uvećani limfni nodusi, desno od 12 mm do 24 mm, pretežno u gornjoj trećini.Supraklavikularno uvećani limfni nodusi do 12 mm.U levoj partiji vrata uvećani i izmenjeni limfni nodusi promera od 14 mm do 35 mm, a supraklavikularno levo do 14 mm.Aksilarno obostrano uvećani i patološki izmenjeni limfni nodusi do 20 mm.U plućnom parenhimu bazalno desno subpleuralni nodus promera do 9 mm, sa trakastim adhezivnim promenama.Slezina heterodenzne strukture, regularnih kontura 143 mm x 59 mm.Jetra regularnih kontura, u AP dijametru u MKL 160 mm, sa promenom u VII segmentu promera 13 mm po tipu hemangioma.Pored opisane promene, uočava se nejasno definisana hipodezna promena promera do 14 mm koja ne pokazuje PKPD.Uočava se i par hipodenznih jasno ograničenih subcentimetarskih promena u V i VI segmentu, bez PKPD, koje po svojim karakteristikama mogu odgovarati cistama. U toku ove hospitalizacije konzilijarno je odlučeno da se kod pacijentkinje započne imunohemioterapijsko lečenje (IHT) po protokolu RFC, D1 I cy 03.12.2019.: Fludarabin, amp.38 mg D1-3, Ciklofosfamid amp.380 mg D1-3, Rituksimab amp.500 mg D4.Primena terapije je protekla bez komplikacija.Otpuštena je kući uz savet da uzima Alopurinol tab. of 15 mm to 27 mm are observed, which are of hypoechoic structures, the rest is unremarkable.Neck, axillary and groin regions US: The size of the thyroid gland is within normal limits, symmetrical with regular contours and homogeneous structure; in the right lobe, heterogeneous cystically degenerated nodule 7.5 mm x 4.5 mm and in the left lobe isoechoic nodule 5 mm x 6 mm.In submandibular area, there is a pair of enlarged LNs, some of which are of altered structure, with a deleted hilus, 10 and 7 mm in diameter.On both sides, parajugularly, several individual LNs are observed in a row, altered structures, erased hili, of preserved L/W index, maximum diameter up to 14 mm.On both sides, in axillae, several grouped enlarged LNs are observed, but with preserved structure and hyperechoic hili, of the preserved L/W index, up to 20 mm in diameter.In both groins, several grouped enlarged LNs, of preserved structure and hyperechoic hili, with preserved L/W index, up to 23 mm in diameter are observed.Chest X-ray findings are within normal limits.CT examination of the neck, chest, and abdomen findings: in the neck region there were enlarged lymph nodes on all levels, on the right-hand side ranging from 12 to 24 mm in diameter, mostly in the upper third.Supraclavicular enlarged lymph nodes were up to 12 mm.On the left-hand side of the neck, there were enlarged and changed lymph nodes with a diameter of 14 to 35 mm, and supraclavicular left up to 14 mm.Axillary bilaterally enlarged and pathologically altered lymph nodes were up to 20 mm.There was a subpleural node in the basal part of the right lung up to 9 mm in diameter, with banded adhesive changes.Spleen was of heterodense structure, regular contours, 143 mm x 59 mm.Liver of regular contours, in AP diameter in MCL 160 mm, with a formation in VII segment, 13 mm in diameter, which 300 mg/dan, Aciklovir i Bactrim 2 x 2 u dane vikenda.Kontrola krvne slike jednom nedeljno.Primenu II cy rituksimaba imala je preko dnevne bolnice KCS 13.01.2020,Primena II cy FC na hematologiji Opšte bolnice u Kraljevu.Primila je na opisani način ukupno 6 cy.Sada se subjektivno dobro oseća, kontrole hematologa na tri meseca.Sadašnja biohemija je u normalnim granicama WBC 4,19 x10 9 /L, RBC 4,12x10 12 /L, HGB 135 g/L. ",
"section_name": "Case report",
"section_num": null
},
{
"section_content": "Hronična limfocitna leukemija je jedna od najčešćih leukemija kod odraslih.Incidencija dostiže 13 obolelih na 100.000 osoba starosti preko 65 godina.Buduči da u početnoj fazi može imati vrlo blage i nespecifične simptome ili čak proticati asimptomatski, potrebne su rutinske analize krvne slike i preventivni pregledi koji su presudni za rano postavljanje dijagnoze i pravovremeno lečenje.Nalaz leukocitoze u krvnoj slici sa limfocitozom je alarm da se pacijent uputi hematologu na dalju dijagnostičku obradu.Takođe, u krvnoj slici može biti prisutna i anemija i/ili trombocitopenija ili hipogamaglobulinemija. Da bi se potvrdila dijagnoza HLL, neophodno je dokazati da su svi limfociti istog porekla (monoklonska limfocitoza).Stoga se vrši njihova imunofenotipizacija (IF), tj.određivanje karakteristika limfocita u perifernoj krvi 3,4 .Kod ćelija HLL uglavnom je dokazano prisustvo markera CD5, CD19, CD23, CD20.Ukoliko se dokaže da su limfociti istog porekla, kao i da ih ima više od 5.000 u uzorku periferne krvi, može se postaviti dijagnoza HLL.Znači, za ranu dijagnozu nisu potrebni invazivni testovi poput punkcije i biopsije koštane srži 5 . Imajući u vidu sporu progresiju bolesti kod većine bolesnika, pre početka lečenja neophodno je odrediti fazu bolesti po Binet i Rai, uraditi imunofenotipizaciju (IF), kao i rizik progresije i prognozu (FISH -Fluorescence in situ hybridization). U toku bolesti može doći do pada imuniteta zbog nefunkcionalnih limfocita, anemije, trombocitopenije, hipogamaglobulinemije što može dovesti do ozbiljnih bakterijskih, virusnih gljivičnih infekcija.Često su kod HLL prisutne hromozomske abnormalnosti, stoga je važno pre otpočinjanja lečenja uraditi FISH analizu kojom se utvrđuje prisustvo specifičnih hromozoma.Del (13 g) -najčešća genetska promena na hromozomu 13, otkriva se kod 40% do 60%, oni imaju povoljniju prognozu, dok Del 17p (deleciju hromozoma 17) ima oko 5% bolesnika i oni imaju najlošiju prognozu, jer ne reaguju na standardne terapije 1,6 . Metodi lečenja su višestruki, a pristup pacijentu individualan.U lečenju se koristi hemioterapija, imunohemioterapija, biološka terapija, radiološka, palijativna, transplantacija koštane srži, opšte mere 5,6 .Hemioterapija se obično sastoji u kombinaciji više lekova.Najčešće se koriste Fludarabin, might be a hemangioma.In addition to the described formation, a vaguely defined hypodense formation up to 14 mm in diameter is observed, which does not show PKPD.A pair of hypodense, clearly limited subcentimeter formations in the V and VI liver segments, without PKPD, can also be observed, and their characteristics may correspond to cysts. During the hospital stay, the Medical Council decided to start immunochemotherapeutic treatment (ICT) according to the RFC protocol, D1 I cy on December 3, 2019: Fludarabine, amp.38 mg D1-3, Cyclophosphamide, amp.380 mg D1-3, Rituximab, amp.500mg D4.There were no complications during the treatment.She was discharged home with the advice to take the Allopurinol tab.300mg /day, Acyclovir and Bactrim, tab.2x2 on weekends.CBC check was advised once a week.The application of II cy of rituximab was performed in the daily clinic of CCS on January 13, 2020, and the application of II cy of FC at Hematology Ward of the General Hospital in Kraljevo.She received a total of 6 cy, as described.Now she feels well and has got regular check-ups with a hematologist every three months.Current CBC is within normal limits WBC 4.19 x10 9 /L, RBC 4.12x10 12 /L, HGB 135 g/L. ",
"section_name": "Diskusija",
"section_num": null
},
{
"section_content": "CLL is one of the most common leukemias in adults.The incidence reaches 13 cases per 100,000 people, over the age of 65.Since in the initial phase it can have very mild and nonspecific symptoms or even be asymptomatic, routine blood counts and preventive examinations are needed, and they are crucial for early diagnosis and timely treatment.The finding of leukocytosis with lymphocytosis is an alarm, and a patient should be referred to a hematologist for further diagnostic evaluation.Anemia and/or thrombocytopenia or hypogammaglobulinemia may also be present in the blood count. In order to confirm the diagnosis of CLL, it is necessary to prove that all lymphocytes are of the same origin (monoclonal lymphocytosis).Therefore, their immunophenotyping (IF) is performed, ie.determination of lymphocyte characteristics in peripheral blood 3,4 .In the CLL cells, the presence of markers CD5, CD19, CD23, CD20 was mainly proven.If it is proven that the lymphocytes are of the same origin, and there are more than 5,000 of them in the peripheral blood sample, a diagnosis of CLL can be confirmed.So, invasive tests such as puncture and bone marrow biopsy are not needed for early diagnosis 5 . Given the slow progression of the disease in most patients, it is necessary to determine the stage of the disease according to Binet and Rai, perform immunophenotyping (IF), as well as the risk of progression and prognosis (FISH -Fluorescence in situ hybridization)., before starting treatment. During the course of the disease, there may be a drop in immunity due to non-functional lymphocytes, anemia, thrombocytopenia, hypogammaglobulinemia, which can lead Ciklofosfamid, Hlorambucil, Bendamustin.Citostatici se mogu davati samostalno ili u kombinaciji sa ciljanom terapijom ili monoklonskim antitelima.Neželjena dejstva koja se mogu javiti su neutropenija, alergijske reakcije, temperatura, mučnina, povraćanje. Imunohemioterapija je kombinacija citostatika i leka koji predstavlja antitelo koje se vezuje za određeni protein na malignoj ćeliji.Ova terapija predstavlja napredak u lečenju, za razliku od hemioterapije koja deluje manje specifično.Terapija monoklonskim antitelima ima za cilj da uništi samo ćelije HLL.Koriste se Rituksimab, Obinutumumab i Ofatumumab 5,6 . Biološka terapija se koristi kod relapsa bolesti, kada bolesnik ne reaguje na klasičnu terapiju, ali i kod pacijenata sa visokorizičnom HLL.Najčešće korišćen lek je Ibrutinib koji blokira Bruton-ovu tirozin kinazu 7,8,9 . Palijativna terapija podrazumeva transfuzije eritrocita i /ili trombocita, primenu antibiotika, kortikosteroida, eventualno operativno vađenje slezine.Transplantacija koštane srži podrazumeva korišćenje matičnih ćelija davaoca.Opšte preporuke podrazumevaju izbegavanje faktora rizika za maligne bolesti, izbegavanje stresa, redovan san, dovoljno tečnosti, čuvati se infekcija i ujeda insekata jer ovi pacijenti imaju intenzivne reakcije na mestu ujeda. Bolesnici sa B ćelijskom HLL ili njenim komplikacijama, prosečno žive 7 do 10 godina.Bolesnici u Rai stepenu od 0-II mogu živeti od 5 do 20 godina.Bolesnici stepena III ili IV umiru za 3 do 4 godine.Napredovanje prema insuficijenciji koštane srži ukazuje na kratko preživljavanje.Bolesnici sa HLL pokazuju sklonost ka drugim malignim tumorima, naročito karcinomu kože.Kako je HLL progresivna bolest neki bolesnici ostaju godinama asimtomatski, pa lečenje nije potrebno pre pojave simptoma ili progresije.Izlečenje je, po pravilu, nemoguće pa je lečenje usmereno na suzbijanje simptoma i produženje života 2 .to serious bacterial, viral, fungal infections.Chromosomal abnormalities are very common in CLL, so it is important to perform a FISH analysis to determine the presence of specific chromosomes before starting treatment.Del (13g) -the most common genetic change on chromosome 13, is detected in 40-60%, and they have a more favorable prognosis, while Del (17p) (deletion of chromosome 17) has about 5% of patients and they have the worst prognosis because they do not respond to standard treatment 1,6 . The treatment methods are multiple, and the patient approach is individual.Chemotherapy, immunochemotherapy, biological therapy, radiological, palliative, bone marrow transplantation, general measures are used in the treatment 5,6 .Chemotherapy usually consists of a combination of several drugs.The most commonly used are Fludarabine, Cyclophosphamide, Chlorambucil, Bendamustine.Cytostatics can be administered alone or in combination with targeted therapy or monoclonal antibodies.Neutropenia, allergic reactions, fever, nausea, vomiting are the most common side effects. Immunochemotherapy is a combination of cytostatics and an antibody that binds to a specific protein on a malignant cell.This therapy represents an improvement in the treatment, unlike chemotherapy which is less specific.Monoclonal antibody therapy aims to destroy only CLL cells.Rituximab, Obinuturumab and Ofatumumab are used 5,6 . Biological therapy is used in the disease relapses, or when the patient does not respond to conventional therapy, but also in patients with high-risk CLL.The most commonly used drug is Ibritunib, which blocks Bruton's tyrosine kinase 7,8,9 . Palliative therapy includes transfusions of erythrocytes and/or platelets, use of antibiotics, corticosteroids, possible surgical removal of the spleen.Bone marrow transplantation involves the use of donor stem cells.General recommendations include avoiding risk factors for malignant diseases, avoiding stress, regular sleep, enough fluids intake, avoiding infections and insect bites because these patients have intense reactions at the site of the bite. Patients with B-cell CLL or its complications live an average of 7-10 years.Patients in Rai grade of 0-II can live from 5-20 years.Grade III or IV patients die within 3-4 years.Progression to bone marrow failure indicates short survival.Patients with CLL are prone to other malignant tumors, especially skin cancer.As CLL is a progressive disease, some patients remain asymptomatic for years, so treatment is not necessary before symptoms or progression occurs.Healing is usually impossible, so treatment is aimed at suppressing symptoms and prolonging life 2 . ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Rutinske analize krvne slike i leukocitarne formule kao i preventivni pregledi od strane izabranog lekara, presudni su u ranom postavljanju dijagnoze HLL.Budići da ova bolest može dugo proticati asimptomatski ili sa blagim i nespecifišnim siptomima, nalaz leukocitoze i limfocitoze u perifernoj krvi je znak da treba misliti na ovo oboljenje i dalju dijagnostiku uraditi u tom pravcu.Poodmakle godine života, muški pol i pozitivna porodična anamneza idu u prilog tome.Diferencijalno dijagnostičke teškoće mogu zadavati infektivna mononukleoza i veliki kašalj.Dalja dijagnostika i lečenje sprovode se na sekundarnom i tercijarnom nivou.Nakon sprovedene specifične terapije, ovi pacijenti bivaju česti posetioci izabranog lekara zbog virusnih, bakterijskih ili gljivičnih infekcija, anemije i drugih posledica lošeg imuniteta. ",
"section_name": "Zaključak",
"section_num": null
},
{
"section_content": "Routine blood counts and leukocyte formulas, as well as preventive check-ups with GP, are crucial in the early diagnosis of CLL.Since this disease can be asymptomatic for a long time or with mild and nonspecific symptoms, the finding of leukocytosis and lymphocytosis in the peripheral blood is a sign that we should take into consideration and perform further diagnostics.Advanced age, male gender, and positive family history are risk factors.Infectious mononucleosis and whooping cough may cause differential diagnostic difficulties.Further diagnosis and treatment are carried out at the secondary and tertiary levels.After specific therapy, these patients become frequent visitors to the GP offices due to viral, bacterial, or fungal infections, anemia, and other consequences of poor immunity. ",
"section_name": "Conclusion",
"section_num": null
}
] |
[] |
10.1371/journal.pone.0002735
|
AIP4/Itch Regulates Notch Receptor Degradation in the Absence of Ligand
|
The regulation of Notch signaling heavily relies on ubiquitination events. Drosophila Su(dx), a member of the HECT family of ubiquitin-ligases, has been described as a negative regulator of Notch signaling, acting on the post-endocytic sorting of Notch. The mammalian ortholog of Su(dx), Itch/AIP4, has been shown to have multiple substrates, including Notch, but the precise events regulated by Itch/AIP4 in the Notch pathway have not been identified yet.Using Itch-/- fibroblasts expressing the Notch1 receptor, we show that Itch is not necessary for Notch activation, but rather for controlling the degradation of Notch in the absence of ligand. Itch is indeed required after the early steps of Notch endocytosis to target it to the lysosomes where it is degraded. Furthermore Itch/AIP4 catalyzes Notch polyubiquitination through unusual K29-linked chains. We also demonstrate that although Notch is associated with Itch/AIP4 in cells, their interaction is not detectable in vitro and thus requires either a post-translational modification, or a bridging factor that remains to be identified.Taken together our results identify a specific step of Notch regulation in the absence of any activation and underline differences between mammalian and Drosophila Notch pathways.
|
[
{
"section_content": "Notch signaling is an evolutionarily conserved process involved in cell fate decisions, cell proliferation or cell death, during development and in the adulthood, and its deregulation leads to several pathologies in mammals.The canonical Notch activation pathway relies on the regulated proteolysis of this membrane receptor after binding to one of its ligands.The resulting free intracellular domain of Notch (ICv) translocates to the nucleus to form a transcriptional activation complex with other cofactors [1].Besides the various combinations of ligands and receptors probably leading to various transcriptional responses, the quantitative aspects of the signaling pathway have to be controlled.These regulations may occur at the level of the production and/or stability of ICv, affinity of the receptor for its ligand, and quantity of ligand and receptor present at the cell surface. Suppressor of deltex (Su(dx)) was first described in Drosophila as a negative regulator of Notch signaling, acting in an antagonist manner to Deltex (dx), another component of the pathway [2][3][4].The phenotypes resulting from the overexpression of the Su(dx) protein in the developing wing were those expected from a downregulation of the Notch pathway.Mammalian orthologs of Su(dx) are called Itch in the mouse and AIP4 in humans.Mammalian Itch was identified in natural mutant mice (itchy mice, [5]) that develop a progressive autoimmune-like disease, partly because Itch targets such as junB are relevant to autoimmunity.However it was recently shown that increased Notch signaling in transgenic mice mimics the symptoms of the disease [6].Various substrates have been described for Itch in mammals: CXCR4 [7], p73, p63 [8,9] smad 7 [10], Jun [11], Deltex (DTX, [12]) and Endophilin [13].In general Itch targets its substrates to degradation, with some exceptions: AIP4 regulates the cell surface expression of select TRP channels by enhancing their ubiquitination and endocytosis but without facilitating their degradation [14].Itch activity on junB is enhanced by Ser/Thr phosphorylation by MEKK1-JNK1 kinases [11], and reduced by Tyr phosphorylation in a fyn-dependent manner [15].Thus it is difficult to attribute general characteristics to Itch, except that it is located in the endosomal system [7,13,16] and that it is autoubiquitinated [12,17].Furthermore the type of chains formed on its substrates is not often identified (except for K29-linked polyubiquitin chains on DTX and itself, [12]).Even less is known in Drosophila about the mechanisms controlled by Su(dx), Notch being its unique described target in this organism.Sakata et al. [18] have shown that ubiquitination of Drosophila Notch depends on Nedd4 (an E3 ubiquitin ligase belonging to the same family as Su(dx)) and on the presence of a PPSY motif in the intracellular region of Notch.Nedd4 is involved in the constitutive endocytosis of Notch and regulates its stability.Wilkin et al. [19] have demonstrated that Su(dx) and/or Nedd4 regulate sorting of Notch full-length within the early endosome.However these authors did not determine whether ubiquitination of Notch targets it for degradation, recycling or some other fate. In mammals, Qiu et al [20] have shown that Itch is able to direct the ubiquitination of Notch DE (a constitutively active but membrane-anchored form of Notch).Based on interaction experiments, they concluded that the Notch IC domain is a direct substrate for Itch.However the step regulated by Itch was not clearly defined. As these conclusions on the role of Itch on an activated form of mammalian Notch contradict the observations made in Drosophila, we decided to identify the form of Notch that was targeted by Itch, and to characterize the consequences of this ubiquitination. We demonstrate here that Itch controls the degradation of the non-activated receptor, inducing after early endocytosis the formation of K29-linked polyubiquitin chains and targeting Notch to lysosomal degradation.We also show that Itch does not interact directly with Notch in mammals and might require a bridging factor. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "It has been shown in Drosophila that Notch signaling is limited by the activity of Su(dx) and DNedd4.In addition the inhibition of Nedd4 activity leads to ligand-independent activation of Notch [18].To test whether the absence of Itch had any consequence on Notch activation in a mammalian system, we transduced MEFs with retroviral vectors to allow expression of human Notch 1 at the cell surface.In this vector an HA tag sequence has been inserted between EGF repeats 22 and 23 of Notch [21].These cells, either WT (called MD-FL) or Itch-/-(ID-FL), have also been stably transfected by a VSV-tagged version of hDTX1 [12].Notch activation was obtained by separation of its heterodimeric form following calcium depletion by EDTA [21].This well documented method allows the rapid and efficient activation of Notch throughout a cell population.Notch activation was monitored by the presence of the Notch intracellular fragment (ICv) in the extracts, detected with the specific V1744 N-terminal antibody (Figure 1).No activated form was detected in the extracts derived from both cell lines, showing that Notch was not constitutively activated in Itch-/-cells (lanes 1 and 4).EDTA treatment led to the production of comparable amounts of ICv in both cell lines (lanes 2 and 5).ICv was also clearly detectable by direct blotting with an anti-Notch IC antibody (compare lanes 8, 11 to 7, 10).EDTA activation of Notch was dependent both on c-secretase activity, since DAPT (a c-secretase inhibitor) treatment abolishes ICv production (lanes 3, 6), and on the membrane metalloprotease TACE, since the S2 cleavage product accumulated in the presence of DAPT (lanes 9, 12, open arrowhead).Thus in the absence of Itch, Notch receptor was fully functional and no ectopic activation was observed.Therefore the increased Notch signaling in Itch -/mice [6] might be rather due to a defect in its negative regulation. ",
"section_name": "Notch activation does not depend on Itch",
"section_num": null
},
{
"section_content": "As the relative quantity of Notch at the membrane was the same in MD-FL and ID-FL cells (Figure S1), we wanted to establish whether Itch regulates the postendocytic sorting of Notch independently of ligand binding.We monitored Notch 1 internalization by an antibody uptake experiment in the absence of any ligand.Notch 1 in MD-FL or ID-FL cells was detected with the use of an extracellular HA epitope (figure 2, 0 min).The cells were incubated with a fluorescently labeled anti-HA antibody at 4uC for 30 minutes, then incubated at 37uC for various periods of time, fixed and observed to track the destination of cell surfacelocalized Notch.Notch receptor incubation with anti-HA antibody did not provoke Notch activation, since no nuclear staining with anti-activated Notch antibody was observed (data not shown).Furthermore the staining pattern, observed with an anti-Notch receptor after fixation and permeabilization, was the same, whether cells had been treated with anti-HA or not (data not shown).After a 30 min incubation at 37uC, much of the Notch immunoreactivity was detected in intracellular vesicles, suggesting that cell surface Notch is constantly internalized in WT as well as in Itch -/-cells (Figure 2, line 30 min).After 60 and 90 minutes, almost no HA staining was detectable in MD-FL cells (first column), suggesting that Notch or the HA antibody had been degraded.Concomitant treatment with the proteasome inhibitor lactacystin did not affect this kinetics, whereas inhibition of lysosomal proteases by leupeptin treatment prolonged the HA staining at least until 90 minutes of internalization (MD-FL+leupeptin).In contrast, in ID-FL cells, HA staining was visible after 60 or 90 minutes of incubation, irrespective of lactacystin or leupeptin treatment (see quantification, figure 2 bottom).On the other hand, EGFR degradation after EGF activation, monitored in the same type of assay with uptake of fluorescent EGF, was identical after a 60 min incubation time in MD-FL and in ID-FL cells (Figure S2, compare MD-FL in A to ID-FL in B).Thus the ID-FL cells are not impaired in a general lysosomal activity or in the endocytic pathway.These results suggest that HA staining disappearance in MD-FL cells is due to Notch degradation in the lysosomes.In ID-FL cells Notch degradation was strongly delayed.Moreover in these cells the Notch-positive endocytic vesicles, detected at 30 minutes (Figure 2) were more scattered throughout the cells, as compared to MD-FL cells.We confirmed these results by co-labelling LAMP-1 after cell fixation and permeabilisation.At 90 minutes of Notch internalization, the HA-positive vesicles in ID-FL were essentially negative for the lysosomal marker LAMP-1 (Figure 3), whether the cells have been treated with leupeptin or not.When MD-FL cells were treated with leupeptin, Notch accumulated in LAMP-1 positive vesicles, showing that it had reached the lysosomes.These results suggest that Notch targeting and degradation in the lysosomes depends on the activity of Itch.To confirm that the presence of Itch is important for this late targeting, we prepared endocytic fractions from MD-FL and ID-FL cells.Notch full-length molecule (indicated as p300 in figure 4A) was detectable in the post-nuclear supernatant (P in lanes 1, 4, 7, 10), and very poorly in the early endosomal fractions (E, lanes 2, 5, 8, 11), confirming the non-contamination of our preparations with Golgi or endoplasmic reticulum proteins [22].This was further verified thanks to an anti-GM130 (a Golgi protein) western blotting (Figure 4B).The heterodimeric receptor, detected as p120 with an antibody recognizing the intracellular part of Notch 1, was present in early endosomal fractions (E) in both MD-FL and ID-FL cells, but in late endosomal fractions (L) only in MD-FL cells.These fractions were enriched in p120 when MD-FL cells were treated with leupeptin, confirming Notch 1 lysosomal degradation (4A, lanes 7 to 9).Itch was mainly detected in E fraction, consistent with its role in this compartment (4B, bottom panel).In ID-FL cells, p120 was only detected in E fractions, irrespective of leupeptin treatment (lanes 5-6 and 11-12).On the other hand, EGFR fractionation was similar in both cell lines (Figure 4B), thus validating the preparations.These results are in agreement with Itch being necessary for Notch transition from early to late endosomes, before lysosomal degradation.To further prove this requirement, we transiently complemented ID-FL cells with AIP4 (Figure 5).Notch expression at the membrane was not affected by overexpression of AIP4 (0 min).On the other hand, Notch degradation was visible after 60 minutes of internalization time in AIP4-transfected cells, whereas in the neighbouring non-transfected cells, Notch was still detected in endocytic vesicles.This result shows that AIP4 complementation is sufficient to restore Notch targeting to lysosomal degradation. ",
"section_name": "Notch receptor in the absence of ligand is targeted to lysosomal degradation in an Itch-dependent manner",
"section_num": null
},
{
"section_content": "In order to determine whether Itch acts on Notch through its E3-ubiquitin ligase activity, we monitored Notch ubiquitination by transfecting 293T cells with expression vectors encoding 66Histagged Ubiquitin together with Notch FL(2CT) (deleted of the C-Figure 3. Notch degradation occurs in the lysosomes.The anti-HA antibody uptake assay was performed as in figure 2. After 90 minutes of chase, cells were fixed, permeabilized and incubated with an anti-LAMP-1 antibody, followed by a mouse-adsorbed rabbit anti-rat and then Al555coupled anti-rabbit antibodies.Insets represent enlargements (fourfold) of the boxed regions.The photographs are representative of a large number of observed fields.The relative amount of Notch-containing vesicles also positive for LAMP-1 was calculated from 10 cells in each condition.In average less than 25% of Notch vesicles were LAMP-1 negative in MD-FL+Leupeptin, whereas less than 25% of Notch vesicles were positive for LAMP-1 in ID-FL cells (+/2 Leupeptin).doi:10.1371/journal.pone.0002735.g003terminal domain, amino-acids 1 to 2183 of Notch1, see [23]), AIP4 or an inactive form of AIP4, carrying a mutation in the active site (named AIP4DN).Ni2+-chelating sepharose was used to purify the ubiquitinated conjugates in denaturing conditions (8M urea), and the ubiquitination levels of Notch were analyzed by immunoblotting.When 66HIS-Ub was coexpressed (Figure 6A, lanes 2-4), we specifically detected a monoubiquitinated Notch species (indicated by *), as well as a smear representing the polyubiquitinated forms (indicated by Ub-Notch).Overexpressing AIP4 enhanced Notch polyubiquitination (lane 3), whereas AIP4DN overexpression severely impaired it (lane 4).To identify the type of isopeptide linkage catalysed on Notch, we made use of expression vectors encoding VSV-tagged ubiquitins, allowing the formation of a single type of polyubiquitin chains [12].After 293T cells transfection with Notch FL(HA) (which contains the carboxyterminal sequence of Notch1), AIP4 and ubiquitin expression vectors, extracts were boiled in SDS-containing buffer, before immunoprecipitation with anti-Notch antibody and detection of the ubiquitinated products with anti-VSV.Ubiquitination of Notch was predominantly detected when using Ub K29, as compared with Ub K48 or K63 (Figure 6B, lanes 2-4).The fact that polyubiquitinated products were detected in these denaturing conditions strongly suggests that they are Notch products.We verified by direct western blotting of the extracts that all ubiquitin vectors allowed the expression and incorporation of the mutant ubiquitins (Figure 6B, bottom).Finally, when monitoring EGFR ubiquitination after EGF treatment, all ubiquitin vectors, including Ub K0 (without any lysine) allowed the same smear to be detected, showing that multiubiquitination was mostly produced, as expected ( [24], Figure S3).This confirmed that preferential K29 ubiquitination was not an artifact of our VSV-Ub vectors.The fact that Notch polyubiquitination was detected with these VSVubiquitin constructs, whereas monoubiquitination was predominant when using His-Ub was an artifact of the His-Ub constructs, that we observed with various ubiquitination substrates and that may be due to steric hindrance caused by the 66His tag.Furthermore the effect of AIP4 WT or DN on Notch (2 or + CT) ubiquitination was also seen with the VSV-ubiquitin expression vectors (Figure S4).However only a mass spectrometry analysis of Notch products would allow the identification of the ubiquitina- tion sites on Notch and of the major type of chains formed.Taken together, our results show that Notch, in the absence of ligand, represents a new substrate of AIP4, submitted to the same modification that we have described for Deltex [12]. In order to precisely identify the step where ubiquitination of Notch by Itch takes place, we monitored Notch FL(2CT) ubiquitination when early endocytosis was inhibited by overexpressing a dominant negative form of rab5 (rab5DN, fused to GFP).As shown in figure 6C, Itch-dependent, K 29-linked polyubiquitination (lanes 5-6) was largely diminished in the presence of rab5DN (lanes 7-8), although Notch quantities were comparable in the extracts (lanes 9-16) and in the immunoprecipitates (lanes 1-8, bottom).We concluded that Itch ubiquitinates Notch after early endocytosis. ",
"section_name": "Ubiquitination of Notch is dependent on Itch",
"section_num": null
},
{
"section_content": "In Drosophila Su(dx) and Notch interact directly through their WW and PPSY motif respectively [18,19,25].The PPSY interaction motif of Drosophila Notch is transformed into PPRL in mammals (aa 2260-2263 in Notch 1), a sequence which is absent in the (2CT) form of Notch.However the presence of the C-terminal domain of hNotch 1 is not necessary for Itch to ubiquitinate Notch (see figure 6A andC).On the other hand Qiu et al [20] have concluded that Itch binds to the N-terminal portion of Notch intracellular domain.We thus examined whether the interaction between Itch and Notch was direct, using GST fusion proteins containing fragments of AIP4 bound to gluthationeagarose beads, and incubated with translation products obtained in rabbit reticulocyte lysates.We used IC forms of Notch (2 and + CT) to avoid the conformation problems due to the presence of transmembrane domains.Figure 7 shows that the IC forms of Notch could not efficiently interact with AIP4 in vitro (lanes 1, 4, 9, 12).Thus a post-translational modification of Notch or Itch might be necessary to allow recognition.Alternatively it is possible that the interaction needs a cofactor.We tested several proteins as possible bridging factors between Notch and AIP4 in this test assay.DTX (two concentrations, indicated with small or large + in lanes 2-7 and 10-15) and numb (lanes 17-22) (see Discussion) were specifically retained on GST-AIP4 in the presence or absence of ICs.In spite of their presence, Notch was not better pulleddown by GST-AIP4 (lanes 5, 6, 13, 14, 21, 22).The same results were obtained when testing b-arrestin 1 or 2 (data not shown). Notch and Itch overexpression and coimmunoprecipitation is not a conclusive assay to demonstrate that Notch interacts with Itch during its trafficking.Hence we decided to purify Notch and Itch-containing complexes from MD-FL cells.We used a large panel of detergents to solubilize membrane proteins, and notably only digitonin allowed specific co-immunoprecipitation of Itch with Notch (Figure 8).This was true when using antibodies directed against either the extracellular part of the receptor (anti-HA, lane 1) or its intracellular domain (Nic, lane 2), suggesting an interaction with the heterodimeric form of Notch.In addition a long exposure of anti-Itch immunoprecipitate immunoblotted with anti-Notch allowed the detection of the p120 form (lane 4 of and a smear corresponding to the ubiquitinated proteins in the whole extracts.C. HEK293T were transfected with expression vectors encoding for Notch FL(2CT), Ub K29, AIP4 (WT) and a dominant-negative form of rab5 linked to GFP (rab5DN) when indicated.Notch products were purified by immunoprecipitation and the ubiquitinated molecules were revealed by anti-VSV (lanes 1-8) followed by anti-Notch (bottom) western blotting.As controls, cell extracts were directly tested for Notch, rab5 (anti-GFP) and Itch (lanes 9-16).doi:10.1371/journal.pone.0002735.g006bottom panel).Taken together, our results suggest that Itch interacts indirectly with heterodimeric Notch in the cells. ",
"section_name": "Interaction between Notch and Itch is not direct",
"section_num": null
},
{
"section_content": "",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Modulating the amount of Notch receptor that is available for signaling could provide one of the mechanisms to finely tune the activity of the pathway.Therefore ubiquitination events and endocytic trafficking of the receptor could constitute key events, particularly in the light of the results showing that mutations that compromise sorting of ubiquitinated membrane protein (ESCRT components for instance) result in overactivation of Notch [26][27][28].Our results show that, in the absence of any activation, mammalian Notch receptor is constitutively internalized and degraded in the lysosomes.Notch degradation in the lysosomes is abolished by treating the cells with leupeptin, whereas a proteasomal inhibitor, lactacystin does not affect this event.Thus the regulation of the quantity of functional Notch receptor occurs mostly through its lysosomal degradation.This result seems contradictory to what was proposed by others, who observed an accumulation of drosophila Notch receptor in the presence of MG101 or MG132 [18,29].However these proteasome inhibitors, unlike lactacystin, could also inhibit lysosomal Cys proteases [30], or they could act indirectly on Notch stability.Recycling of Notch receptor is not completely ruled out, in particular in the Itch -/cells, but this type of event should have been detected in our antibody uptake experiment where the antibody used did not dissociate from the receptor in a moderately acidic environment. We show here that the initial steps of Notch endocytosis do not depend on Itch/AIP4.However this E3 ubiquitin ligase is required at a later step allowing final targeting to the lysosomes, since we observe that Notch is still internalized in Itch-/-cells and accumulates in endosomes that are adjacent to LAMP-1 positive vesicles and that ubiquitination is impaired when early endocytosis is blocked.It should be mentioned that although Notch 35 S met, and their ability to be retained onto a GST-AIP4 fusion protein or control GST adsorbed to glutathione-agarose beads was analyzed by SDS-PAGE analysis.The input lanes (7,8,15,16,23,24,25) show the different in vitro-translated products prior to incubation with the beads.Two concentrations of in vitro translated DTX (0.5 or 3 ml, indicated by + and + respectively) were tested.White lines indicate that intervening lanes have been spliced out.doi:10.1371/journal.pone.0002735.g007degradation is severely delayed in Itch -/-cells, other members of the Itch family such as Nedd4 are still present in these cells and may partially replace it.Nevertheless the reintroduction of AIP4 in ID-FL cells is required to fully restore the kinetics of Notch degradation.We also propose that AIP4/Itch mainly catalyses the formation of polyubiquitin chains through the lysine 29 of ubiquitin, as is the case for two of its substrates, DTX and itself [12].Hence Notch seems to behave like DTX towards AIP4, both regarding the type of isopeptide linkage used and the localisation and time of the ubiquitination events in the endocytic pathway. It should be mentioned that other ubiquitination events affecting Notch and catalyzed by other E3 ubiquitin ligases could happen before or after Itch requirement.The RING E3 ubiquitin ligase c-cbl, which was described to be necessary for Notch lysosomal degradation [31] could fulfill this function.On the other hand, other receptors have been recently described to be endocytosed without any ubiquitination event, this step taking place later.This was recently demonstrated for EGFR, where mutating all lysine residues in the kinase domain did not affect the internalization rate of the receptors [32].More complicated situations may be envisaged, as recently shown for the interferon receptor IFNAR1, where ubiquitinations through K48 and K63 linkages on specific sites are required for the receptor to be internalized after ligand stimulation, the efficiency of this process being affected by the ubiquitination-dependent exposure of a linear endocytic motif present in the cytoplasmic tail of the receptor [33].Therefore mechanisms underlying Notch receptor endocytosis are not completely understood yet, although its ubiquitination through K29 chains represents a signature of a very specific Itch-dependent spatiotemporal step. ",
"section_name": "Role of Itch/AIP4 in Notch signaling",
"section_num": null
},
{
"section_content": "The Drosophila Su(dx) gene was described as a negative regulator of Notch signaling [2][3][4].It has been shown that Su(dx) and Nedd4 regulate the postendocytic sorting of Notch en route to the late endosome [18,19].Our data confirm and extend these observations by identifying the way Notch is degraded and the step regulated by Itch.Nedd4 suppression in drosophila leads to Notch-dependent and ligand-independent activation of Notch target genes [18], possibly by stabilizing the Notch-Dx complex and facilitating c-secretase proteolytic cleavage of Notch.We did not observe any constitutive activation of Notch in Itch -/-cells, although Notch was fully activable by EDTA treatment even in the absence of Itch.This discrepancy might be related to the different protein complexes formed in drosophila and in mammals.Indeed it has been well documented that Su(dx) WW domains directly interact with a PPSY motif in the drosophila Notch intracellular domain [18,19,25].Our data using GST pulldown experiments show that unmodified forms of Notch or AIP4 cannot stably interact together.Moreover the crucial tyrosine present in the interaction motif of Drosophila Notch is absent in mammalian Notch, and the presence of the C-terminal part of mammalian Notch does not affect its ability to be ubiquitinated by Itch.Taken together, these results suggest that mammalian Notch does not use such a motif to interact with Itch.Therefore the protein complexes containing Notch and Su(dx) in drosophila might be different from those containing Notch and Itch/AIP4 and found during endocytosis in mammals. Qiu et al [20] concluded that murine Itch binds to the Nterminal portion of Notch intracellular domain, although they did not prove that the interaction was direct.We were able to coimmunoprecipitate endogenous Notch and Itch only when solubilizing membrane complexes with the mild detergent digitonine.Therefore such membrane Notch-containing complexes might also contain other proteins, the presence of which is necessary to connect Notch and Itch.The same type of events have been described in mammals for other HECT-E3 ubiquitin ligases.In particular after TGFb stimulation, Smurf1 and Smurf2 participate in the ubiquitination and degradation of TGFb receptor and SnoN, through their association with Smad7 and Smad2 respectively [34][35][36]. Based on genetical and biochemical data, we tested in vitro two possible bridging factors between Itch and Notch, DTX and numb.DTX in mammals has been shown to interact with Itch [12] and Notch [37], and is associated with endocytic vesicles [12].Numb was proposed to promote Notch receptor ubiquitination and degradation of its intracellular domain after activation [38].On the other hand, numb targets Gli1 for Itch-dependent ubiquitination [39].Although both proteins could constitute good candidates, their presence did not improve Notch binding to GST-AIP4.Finally we tested beta-arrestin 1 and 2, since b-arrestin 1 was very recently demonstrated to be necessary to promote the interaction between AIP4 and activated CXCR4 [40].Furthermore b-arrestin 2 drosophila ortholog, Krz, was described as a negative regulator of Notch signaling acting in a deltex-dependent manner [29].However these factors did not facilitate Notch binding to AIP4 in vitro.Therefore we conclude that either other What happens when Notch is activated? Our results show that activation of Notch FL is not affected by the presence of Itch.However both types of fates, Itch-dependent Notch degradation on one hand, ligand-dependent Notch activation on the other hand, rely on endocytosis of the receptor [41].One can imagine two possibilities: in the first model, Itch is associated with Notch complexes at the membrane, then ligand activation and ADAM cleavage produce a new form which can no longer interact with Itch, thus allowing Notch sorting to different vesicles where the interaction with c-secretase elements and subsequent cleavage can occur.Alternatively Itch association occurs after sorting of activated Notch to the vesicles where it is cleaved by the c-secretase complex, and thus can happen only by default on the receptor molecules which have not been activated.This hypothesis is however difficult to reconcile with the fact that Itch colocalizes with Hrs-positive vesicles [7,12,40] and that csecretase cleavage seems to occur downstream of these vesicles [41][42][43].Irrespective of the model, further experiments are required to characterize the discriminating events and the partners involved in the sorting.Transfections: Ca-phosphate for HEK293T, Fugene 6 (Roche) for Plat-E, Fugene HD (Roche) for MEF cell lines, according to manufacturer's instructions. ",
"section_name": "Parallels and Differences between drosophila Su(dx) and mammalian Itch/AIP4",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "Cell cultures, immunofluorescence, immunoprecipitations and immunoblots experiments were performed as previously described [12].For immunofluorescence, images were acquired with 0,3 mm sections using an Axioplan 2 imaging with Apotome system (Carl Zeiss MicroImaging Inc., Le Pecq, France).The magnification for all photographs was 636. ",
"section_name": "Reagents, constructs and cellular biochemical studies",
"section_num": null
},
{
"section_content": "Ni sepharose purification: 293T cells were harvested 24 h after transfection and lysed in 8 M urea, 0.1 M NaH2PO4, 10 mM Tris-Hcl (pH 8), 1% Triton X-100 and 20 mM Imidazole at room temperature.His-Ub conjugated proteins were purified on chelating Sepharose beads (Pharmacia), prealably charged with Nickel.Ni-bound proteins were washed extensively with the same buffer, then with a pH 6.3 buffer and eluted in Laemmli before western blot analysis. Denaturing immunoprecipitation: 293T cells were collected 24 h after transfection, washed in PBS buffer and lysed in 50 mM Tris-Hcl (pH 8.0), 1% Triton X-100, 150 mM NaCl supplemented with 16protease inhibitor cocktail (Roche) and 5 mM Nethylmaleimide (Sigma).After removing of the insoluble material by centrifugation, extracts were boiled in 1% SDS for 5 minutes.After neutralization of SDS by 10-fold dilution in Triton buffer, immunoprecipitation was performed. ",
"section_name": "Ubiquitin-conjugates purification",
"section_num": null
},
{
"section_content": "After washing the cells in serum-free medium, they were incubated at 4uC for 30 min with anti-HA-Al488 (or a mixture with EGF-Al555), then washed again and incubated in serum-free medium for various periods of time at 37uC.The cells were then quickly rinsed in cold PBS, fixed and processed for immunostaining.If stained additionally with anti-LAMP-1, this was applied after the permeabilization step. ",
"section_name": "HA antibody or EGF uptake assays",
"section_num": null
},
{
"section_content": "The preparation of early and late endosomal enriched fractions from MD-FL or ID-FL cells was performed after 16 h of leupeptin treatment according to [44].Briefly, cells were washed and harvested in ice-cold PBS, pelleted and resuspended in 250 mM Sucrose, 20 mM Tris pH 7.9, 3 mM Imidazole.Cells were broken with a dounce (pestle B), the post nuclear supernatant (P) was collected after centrifugation at 3000 rpm for 10 min.It was then brought to 40.6% sucrose, loaded at the bottom of an ultracentrifuge tube, and sequentially overlaid with 35%, 25% and 8.6% sucrose containing buffers.The gradient was centrifuged at 35000 rpm for one hour at 4uC in a SW55Ti rotor.Early and late endosomal fractions were collected at the 35/25 and 25/8.6%sucrose interfaces respectively. ",
"section_name": "Suborganellar fractionation",
"section_num": null
},
{
"section_content": "Notch heterodimer dissociation was obtained as described [21].After washing with HBSS (Gibco-BRL), the cells were incubated in pre-warmed 10 mM EDTA-containing HBSS for 15 min at 37uC.The medium was replaced by PBS and cells were directly collected and lysed.When necessary, DAPT (5 mM) was added 1 h before activation and maintained during the treatments. ",
"section_name": "Activation by EDTA treatment",
"section_num": null
},
{
"section_content": "In vitro-translated proteins were synthesized in a reticulocyte lysate-coupled transcription/translation system (Promega), in the presence of 35 S methionine.Approximately equal amounts of glutathione-S transferase (GST) alone or in fusion with AIP4 [17], as estimated from a Coomassie-stained gel, were bound to glutathione-Agarose (Sigma).The in vitro translations were incubated for 2 hours at 4uC with these beads in 1% Tritoncontaining buffer.The beads were then extensively washed in the same buffer and the bound proteins were eluted by boiling in Laemmli buffer, subjected to SDS-PAGE analysis followed by fluorography. ",
"section_name": "GST pull-down analysis",
"section_num": null
},
{
"section_content": "Figure S1 MD-FL and ID-FL cells exhibit similar amounts of Notch at the membrane: Surface proteins of both cell lines were labelled with NHS-biotin for 1 hour at 4uC (lanes +).Whole cell extracts (lanes 1-4) and fractions retained on streptavidin-agarose (lanes 5-8) were analyzed by immunoblotting with anti-Notch.Note that only p120 is detected in lanes 6 and 8, confirming the non-contamination with intracellular Notch.As a positive control, EGFR was detected at the membrane, and GM130 was used as a negative control.Immunoreactivity of the upper blot was quantified using Quantity One software (Biorad Lab.).For each cell line, the relative amount of membrane Notch was calculated as the ratio between «streptavidin-purified» and «extract» signals.It was estimated to 3.5% in MD-FL cells and 3.3% in ID-FL cells.6B).Ub K0 indicates a mutant where all lysine residues have been replaced by arginine.24 hours after transfection, cells were treated with EGF for 5 minutes, extracts were prepared and immunoprecipitated with a mouse anti-EGFR (Santa Cruz sc-120).Ubiquitinated products were revealed by western blotting with anti-VSV antibody and controlled by western blotting with rabbit anti-EGFR (sc-03).A similar smear was observed with all ubiquitin constructs, even K0, in accordance with published data showing that multiubiquitination was mostly produced after EGFR activation. ",
"section_name": "Supporting Information",
"section_num": null
}
] |
[
{
"section_content": "We thank S. Conner, R. Kopan, M. Zerial, M. Treier, J. Aster, A. Angers, A. Atfi for generous gifts of materials, F. Logeat for helpful discussions and critical reading of the manuscript. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "This project was supported in part by grants from Association pour la Recherche sur le Cancer (#4840) to C. Brou, and from the European Community (# 018683, NoE RUBICON) to A. Israe ¨l. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Conceived and designed the experiments: PC CB.Performed the experiments: PC CB.Analyzed the data: PC CB.Wrote the paper: AI CB. ",
"section_name": "Author Contributions",
"section_num": null
}
] |
10.3390/ijms24065728
|
Physiological and Pathophysiological Roles of IgM Fc Receptor (FcµR) Isoforms
|
<jats:p>IgM is the first antibody to emerge during phylogeny, ontogeny, and immune responses and serves as a first line of defense. Effector proteins interacting with the Fc portion of IgM, such as complement and its receptors, have been extensively studied for their functions. IgM Fc receptor (FcµR), identified in 2009, is the newest member of the FcR family and is intriguingly expressed by lymphocytes only, suggesting the existence of distinct functions as compared to the FcRs for switched Ig isotypes, which are expressed by various immune and non-hematopoietic cells as central mediators of antibody-triggered responses by coupling the adaptive and innate immune responses. Results from FcµR-deficient mice suggest a regulatory function of FcµR in B cell tolerance, as evidenced by their propensity to produce autoantibodies of both IgM and IgG isotypes. In this article, we discuss conflicting views about the cellular distribution and potential functions of FcµR. The signaling function of the Ig-tail tyrosine-like motif in the FcµR cytoplasmic domain is now formally shown by substitutional experiments with the IgG2 B cell receptor. The potential adaptor protein associating with FcµR and the potential cleavage of its C-terminal cytoplasmic tail after IgM binding are still enigmatic. Critical amino acid residues in the Ig-like domain of FcµR for interacting with the IgM Cµ4 domain and the mode of interaction are now defined by crystallographic and cryo-electron microscopic analyses. Some discrepancies on these interactions are discussed. Finally, elevated levels of a soluble FcµR isoform in serum samples are described as the consequence of persistent B cell receptor stimulation, as seen in chronic lymphocytic leukemia and probably in antibody-mediated autoimmune disorders.</jats:p>
|
[
{
"section_content": "Two separate lineages of lymphocytes are generated in their distinctive tissue sites for adaptive immunity.B cells are developed in the bone marrow in mammals and the bursa of Fabricius in chickens and contribute to humoral immunity, whereas T cells are generated within the thymus and contribute to cellular immunity [1].Antibody or immunoglobulin (Ig) is a key player in the humoral immunity and has a dual-binding activity: usually first to antigens via its two identical N-terminal variable domains in the Fab region and then to effector molecules via its constant region in the Fc region (except for IgE-FcεRI interaction).Of five different antibody classes, IgM is the first antibody class to appear during phylogeny (from jawed vertebrate onward), ontogeny (from the 8th-9th week of gestation in humans), and in immune responses (a few days after antigen exposure).On the surface of B cells, monomeric IgM is part of the B cell antigen receptor (BCR) [2], and at their terminally differentiated plasma cells, secreted pentameric IgM initiates for humoral immunity.It is evident from studies of mice unable to secrete IgM that both pre-immune \"natural\" and antigen-induced \"immune\" IgM are important for protective immunity and regulation of autoimmune processes by recognizing pathogens and self-antigens [3,4].Effector molecules interacting with the Fc region of IgM, such as complement and its receptors, have thus far been extensively studied for IgM-mediated protection and immune regulation [5,6].In contrast, studies investigating the role of the IgM Fc receptor (FcµR) in IgM effector functions have been limited, which is in part due to its identification only ~14 years ago [7].Since many review articles on FcµR have already been published elsewhere [4,[8][9][10][11][12][13], this article briefly reiterates the unique features and unresolved issues of FcµR and discusses contradicting views on various aspects of this receptor. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "Most of the available evidence indicates that unlike FcRs for switched Ig isotypes, which are expressed by a variety of immune and non-hematopoietic cell types [13,14], FcµR is more selectively expressed by B cells, T cells, and, to a lesser extent, NK cells in humans, and only B cells in mice.This conclusion is based on various analyses: (i) flow cytometric assessments using IgM ligands and FcµR-specific monoclonal antibodies (mAbs) to stain both freshly prepared primary cells and established cell lines before and after activation with various stimuli ex vivo, (ii) biochemical characterization of proteins isolated from cellular lysates using both IgM ligands and mAbs, and (iii) sensitive reverse transcription polymerase chain reaction (RT-PCR) assays detecting FcµR transcripts [7,9,10,13,15].However, a considerable number of papers have also described the expression of FcµR by non-B cells in mice (i.e., granulocytes, monocyte/macrophages, dendritic cells (DCs), and Th17 cells), mainly based on the functional alterations in these cell types in a particular strain of the Fcmr-deficient (KO) mouse line (designated as Fcmr KO-M in this article) [16][17][18]. Recently, Kubli et al. described that FcµR negatively regulated anti-tumor activity by tumor-associated mononuclear phagocytes (TMPs) in a melanoma mouse model using the Fcmr KO-M strain.The mutant mice had increased numbers of TMPs, a reduced tumor size, and showed improved survival as compared with control wildtype (WT) mice.Single-cell RNA sequence (scRNAseq) analysis of TMPs from Fcmr KO-M and WT mice revealed a unique TMP subset with enhanced antigen processing/presenting properties in the mutant mice [19].However, when we reexamined the scRNAseq data of TMPs, we found no Fcmr expression by such TMPs [20].Almost none of the analyzed TMPs (8000 Fcmr KO-M and 6352 Fcmr WT cells) had Fcmr transcript reads.Notably, one WT cell with four Fcmr transcript reads was found to also contain transcript detection of B cell-specific genes.These results are consistent with the findings from the comprehensive scRNAseq analyses of various mononuclear phagocytic populations conducted by Hume and his colleagues, showing that Fcmr transcripts were a marker for a B cell-specific contaminant cluster [21,22].In reply to our aforementioned comments [20], Kubli et al. assumed a technical limitation as a reason for the lack of Fcmr transcripts in TMPs, a so-called dropout effect, and emphasized on the surface-staining data of FcµR expressed on myeloid cell as determined by a 'novel' rat antiserum, instead of mAb, which was raised against a recombinant soluble FcµR protein [23].Unfortunately, there was no demonstration of the specificity of such polyclonal antiserum by immunoprecipitation and proteomic analyses.Thus, there is no solid evidence for the expression of FcµR by myeloid cells, including TMPs (see Figure 1A).It is also noteworthy that many kinds of artifacts may occur in scRNAseq analyses, including the fragmentation of tissue-resident macrophages during isolation, contamination, stochastic sampling, and even non-zero sequencing of DNA fragments [21,22].Retrospectively, it seems likely that the functional alterations observed in non-B cell populations of this particular Fcmr KO-M mouse resulted from an off-target effect or the consequence of the Fcmr ablation procedures unrelated to the inactivated Fcmr itself (see below). effect or the consequence of the Fcmr ablation procedures unrelated to the inactivated Fcmr itself (see below). ",
"section_name": "Disputes on the Cellular Distribution of FcµR 2.1. Questioning Whether FcµR Is Expressed by Mouse Innate Immune Cells",
"section_num": "2."
},
{
"section_content": "Microfold (M) cells reside in the follicle-associated epithelium at Peyer's patches and have a unique function to deliver a variety of materials, including particulate antigens, soluble macromolecules, and pathogens from the intestinal lumen to Peyer's patches [24,25].In addition to sampling of these heterogenous materials by M cells, immune complexes of secretory IgA (SIgA) and antigens in intestinal lumens have been shown to be reversely transcytosed through the mucosal epithelium by selective binding to receptors, Dectin-1 (C-type lectin) and Siglec-5 (sialic acid binding Ig-like lectin 5), expressed on the apical surface of M cells [26].Recently, the same group of authors extended this receptormediated, reverse transcytosis to secretory IgM (SIgM) and found that FcµR was expressed on the apical surface of M cells and was involved in the M cell-mediated reverse transcytosis of SIgM/antigen complexes, suggesting a regulatory role of FcµR in mucosal immunity [27] (see Figure 1B).However, IgM can bind various proteins other than FcµR (e.g., CD22/siglec-2, tripartite motif-containing protein 21 (TRIM21)/E3 ubiquitin ligase, apoptosis inhibitor of macrophages (AIM)/soluble protein α/CD5L, Fcα/µR, polymeric Ig receptor (pIgR), mannan binding protein, binding Ig protein (Bip)/heat shock protein A5 (HSPA5)/78 kDa glucose-regulated protein (GRP78)).Moreover, preliminary findings in ",
"section_name": "Reverse Transcytosis of Secretory IgM via FcµR Expressed on M Cells in Peyer's Patches",
"section_num": "2.2."
},
{
"section_content": "Microfold (M) cells reside in the follicle-associated epithelium at Peyer's patches and have a unique function to deliver a variety of materials, including particulate antigens, soluble macromolecules, and pathogens from the intestinal lumen to Peyer's patches [24,25].In addition to sampling of these heterogenous materials by M cells, immune complexes of secretory IgA (SIgA) and antigens in intestinal lumens have been shown to be reversely transcytosed through the mucosal epithelium by selective binding to receptors, Dectin-1 (C-type lectin) and Siglec-5 (sialic acid binding Ig-like lectin 5), expressed on the apical surface of M cells [26].Recently, the same group of authors extended this receptor-mediated, reverse transcytosis to secretory IgM (SIgM) and found that FcµR was expressed on the apical surface of M cells and was involved in the M cell-mediated reverse transcytosis of SIgM/antigen complexes, suggesting a regulatory role of FcµR in mucosal immunity [27] (see Figure 1B).However, IgM can bind various proteins other than FcµR (e.g., CD22/siglec-2, tripartite motif-containing protein 21 (TRIM21)/E3 ubiquitin ligase, apoptosis inhibitor of macrophages (AIM)/soluble protein α/CD5L, Fcα/µR, polymeric Ig receptor (pIgR), mannan binding protein, binding Ig protein (Bip)/heat shock protein A5 (HSPA5)/78 kDa glucose-regulated protein (GRP78)).Moreover, preliminary findings in collaboration with Takashi Kanaya and Hiroshi Ohno (RIKEN, Yokohama, Japan; unpublished) have raised the question of whether FcµR is indeed expressed by M cells in follicle-associated intestinal epithelium.Thus, the validity of the above findings of M cell-mediated reverse transcytosis of SIgM via FcµR must await further confirmation. ",
"section_name": "Reverse Transcytosis of Secretory IgM via FcµR Expressed on M Cells in Peyer's Patches",
"section_num": "2.2."
},
{
"section_content": "So far, five different strains of Fcmr KO mice have been developed by four different laboratories using different targeting strategies, and at least eight different groups of investigators have examined their resultant phenotypes, with some discrepancies (see reviews [12,13]).However, the common finding among these mutant mice is an impairment of B cell tolerance, as evidenced by enhanced serum titers of autoantibodies of both IgM and IgG [12,13] (Figure 2).The major discrepancy among the reported phenotypes is mainly attributed to the functional alterations of non-B cell populations observed in the Fcmr KO-M strain [13].The Fcmr KO-M mice are unique among all Fcmr KO strains in their extensive deletion of genomic DNA (~10 kb), ranging from exon 2 (signal peptide-2 and Ig-like domain) to exon 8 (cytoplasmic tail-3 and 3 untranslated region).Moreover, the Neo resistance cassette remained in the mouse genome [16], a situation that is known to impact on mouse phenotypes.According to the epigenetic analysis of the Fcmr-Il10 locus in regulatory T (Treg) cells by Ohkura and Sakaguchi [28], three loci, the 3 region of Fcmr (exon 5 (transmembrane) to exon 8), and 5 upstream of Il10 and Il10, were selectively in an open chromatin configuration when Treg cells were activated; hence, such loci might be highly accessible to transcription factors [13].If this selective epigenetic alteration seen in Treg cells is also the case for myeloid cells including TMPs, it may account for different myeloid functions in the Fcmr KO-M strain (missing this 3 region of Fcmr) as compared with its WT control and other Fcmr KO strains.Supporting this, a difference in granulocyte function was noted between Fcmr KO-M and our KO (which lack exons 2-4 [29]) mice.Production of reactive oxygen species was significantly higher in the former KO granulocytes than WT controls upon stimulation with N-formylmethionine-leucylphenylalanine in the presence or absence of lipopolysaccharide (see Figure 3C in [16]), but was comparable in our Fcmr KO and WT granulocytes [20].Collectively, any alterations in non-B cell functions observed in the Fcmr KO-M strain [16][17][18] most likely are not due to the lack of FcµR per se but to an off-target effect caused by the extensive targeting strategy as compared to that of other Fcmr mutant strains. ",
"section_name": "Common and Conflicting Findings in Fcmr-Deficient Mice",
"section_num": "3."
},
{
"section_content": "FcµR is encoded by a single copy gene located on chromosome 1q32.2adjacent to those encoding two promiscuous IgM-binding receptors, pIgR and Fcα/µR.FcµR is a type I transmembrane sialoglycoprotein with an M r of ~60 kDa, which consists of a V-set Ig-like domain responsible for exclusive Fcµ binding, an additional extracellular (EC) stalk region, a transmembrane (TM) segment containing a charged His residue at position 253 (H 253 ), and a relatively long cytoplasmic portion containing evolutionary conserved, three Tyr, and five Ser residues [7].Among these Tyr residues, the C-terminal Y 385 matches the Ig-tail Tyr (ITT) motif (DYxN, where x indicates any amino acid (aa)) seen in the cytoplasmic tails of membrane-bound IgG and IgE [31,32].The two C-terminal Tyr residues Y 366 and Y 385 of FcµR were found to be involved in FcµR-mediated endocytosis [33,34], whereas the membrane proximal Y 315 was involved in the receptor-mediated protection from agonistic IgM anti-Fas mAb-induced apoptosis in vitro [34].When the fate of IgM bound to FcµR on stable transductants expressing WT or H253F (a point mutation of His at position 253 to Phe) mutant form was examined by immunofluorescence microscopy, the mutant showed enhanced cap formation even at 4 • C, suggesting an anchoring role of the H 253 residue of FcµR in the plasma membrane [34].Since we have so far successfully developed FcµR (membrane form) stable transductants using various lymphoid cell lines as hosts, we initially considered that, unlike activating isoforms of paired receptors, FcµR could be displayed on the plasma membrane without an adaptor protein that carries a negatively charged aa residue in its TM to neutralize the H 253 residue of FcµR.Alternatively, the relatively long cytoplasmic part of FcµR might compensate for such adaptor-dependent surface expression.However, when murine 3T3 fibroblasts were transduced with a retroviral bicistronic expression vector encoding FcµR and green fluorescein protein (GFP) cDNAs, the resultant GFP + cells did not express FcµR on their cell surface, suggesting the requirement of companion molecules, not present in fibroblasts, for FcµR to reach the plasma membrane (Marie Burns and HK, unpublished).This scenario is reminiscent of our findings with paired Ig-like receptors (PIRs) that the FcR common γ chain is a prerequisite for the cell surface expression of PIR-A (short cytoplasmic tail, activating isoform), but not of PIR-B (long cytoplasmic tail, inhibitory isoform), in fibroblasts [35].Whether or not such a companion molecule (e.g., an adaptor protein non-covalently associating with FcµR) exists remains to be elucidated (Figure 3A).Another unresolved issue is the enhanced migration of phosphorylated FcµR on sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) upon receptor ligation or treatment of FcµR-expressing cells with the tyrosine phosphatase inhibitor pervanadate [7].Pronounced phosphorylation of FcµR at both Tyr and Ser residues might cause a global structural change, leading to faster migration on SDS-PAGE, unlike the usually observed slower migration.Alternatively, it might be due to proteolytic cleavage of FcµR, after receptor ligation.Preliminary findings with epitope-tagged FcµR transductants suggested a potential cleavage of its cytoplasmic tail (Eugene J. Becker, Yoshiki Kubagawa, Marie Burns, and HK, unpublished).In this regard, ligation of the FcγRIIa, an activating FcγR, on platelets leads to activation of both the metalloprotease that targets the collagen receptor glycoprotein VI to shed its ectodomain and the intracellular calpain that cleaves the cytoplasmic tail of FcγRIIa to remove the ITAM-containing stub.This finding suggests a potentially novel mechanism for platelet dysfunction by FcγRIIa after immunological insult, including IgG autoantibodies to platelets [36].The precise molecular basis for the enhanced migration of phosphorylated FcµR awaits further investigation (Figure 3B). Collectively, unlike pairs of activating and inhibitory receptors, FcµR itself may possess a dual-signaling ability.One results from a putative ITAM-bearing adaptor protein, which may non-covalently associate with FcµR via H 253 , and another one involves Tyr and/or Ser residues within the cytoplasmic FcµR domain. ",
"section_name": "Structural and Signaling Aspects of FcµR 4.1. Dual-Signaling Potential of FcµR",
"section_num": "4."
},
{
"section_content": "To determine if the ITT-like motif (DY 385 INV) in FcµR indeed has the capacity to transduce intracellular signaling, we tested if the cytoplasmic tail of FcµR can functionally compensate for that of membrane-bound IgG (mIgG).To this end, we replaced the cytoplasmic domain of mIgG2a with that of FcµR and expressed the resulting chimeric BCR in the IgM-bearing human B cell line DG75 (mIgG2a/FcµR WT).As a control, we inactivated the FcµR ITT-like motif by replacing the central Tyr at position 385 with phenylalanine (mIgG2a/FcµR Y385F) (hereafter, mIgG2a/FcµR WT and mIgG2a/FcµR Y385F are designated as WT and YF, respectively, for simplicity).Both WT and YF stable transductants expressed similar levels of mIgG2a-BCRs on their cell surfaces.To test the signaling capacities of the chimeric mIgG-BCRs, we analyzed mobilization of the second messenger Ca 2+ on BCR activation.Ligation of the chimeric mIgG2a-BCRs induced significantly stronger Ca 2+ mobilization in the WT than in the YF mutant transductants, suggesting a signal-amplifying function of the ITT-like motif of FcµR, similar to that of wildtype mIgG-BCRs (Figure 4A).Importantly, the different Ca 2+ mobilization kinetics were not due to cell-intrinsic variations since stimulation of endogenous mIgM-BCRs gave rise to similar Ca 2+ mobilization profiles in both transductants, of which the surface IgM levels were indistinguishable.Western blot analyses revealed that chimeric mIgG2a/FcµR protein of 62 kDa was tyrosine-phosphorylated post-ligation in both WT and YF transductants.These results indicate that while the ITT-like motif of FcµR can functionally replace the ITT of mIgG2a-BCRs, there are additional tyrosine residues in the FcµR cytoplasmic domain that undergo phosphorylation when brought into the immediate proximity of an activated BCR.However, only the ITT-like motif seems to interact with the adaptor protein Grb2, since it was co-precipitated with ITT-WT but not -YF mutant mIgG2a/FcµR fusion proteins. The functional core of ITT-like motifs consists of the aa sequence YxN [32].To investigate the signaling impact of aa that flank the FcµR ITT-like motif in more detail, we replaced the aa surrounding the ITT core motif of mIgG2a with those of the FcµR (Figure 4B).The resulting mIgG2a-ITT (FcµR) chimeric variants contained the modified ITT sequence SDDYINVPGQ or the phosphorylation-deficient sequence SDDFINVPGQ (hereafter referred to as mIgG2a-ITT (FcµR) WT or YF, respectively).These constructs (Figure 4C, right), as well as mIgG2a ITT WT or YF mutants as controls (Figure 4C, left), were expressed and analyzed in DG75 B cells, as above.The resultant transductants expressed very similar levels of mIgG2a-BCRs on their cell surfaces.In both cases, stimulation of the WT versions of mIgG2a-BCRs gave rise to much stronger Ca 2+ mobilization profiles than in cells expressing the YF mutant mIgG2a-BCR variants (Figure 4C).As before, stimulation of endogenous mIgM-BCRs confirmed that these differences were not due to cell-intrinsic variations.Notably, anti-phosphotyrosine Western blot analysis of purified mIgG2a-BCRs showed a much stronger ITT phospho-signal, along with enhanced co-precipitation of Grb2 in cells expressing the modified mIgG2a-ITT (FcµR) BCR as compared to cells expressing the WT mIgG2a-ITT BCR.These results indicate a more efficient phosphorylation of the FcµR ITT-like motif than that of the mIgG2a ITT, which goes along with its more potent Grb2 recruiting activity.This improved ITT phosphorylation may be caused by the additional Asp residue in the minus 2 position relative to the Tyr.However, it does not seem to further enhance amplification of Ca 2+ mobilization.since it was co-precipitated with ITT-WT but not -YF mutant mIgG2a/FcµR fusion proteins.The functional core of ITT-like motifs consists of the aa sequence YxN [32].To investigate the signaling impact of aa that flank the FcµR ITT-like motif in more detail, we replaced the aa surrounding the ITT core motif of mIgG2a with those of the FcµR (Figure 4B).The resulting mIgG2a-ITT (FcµR) chimeric variants contained the modified ITT sequence SDDYINVPGQ or the phosphorylation-deficient sequence SDDFINVPGQ (hereafter referred to as mIgG2a-ITT (FcµR) WT or YF, respectively).These constructs (Figure 4C, right), as well as mIgG2a ITT WT or YF mutants as controls (Figure 4C, left), were expressed and analyzed in DG75 B cells, as above.The resultant transductants expressed very similar levels of mIgG2a-BCRs on their cell surfaces.In both cases, stimulation of the WT versions of mIgG2a-BCRs gave rise to much stronger Ca 2+ mobilization profiles than Collectively, these findings clearly demonstrate that the ITT-like motif of FcµR has the ability to mimic the signaling function of the mIgG2a ITT.The amino acids surrounding the FcµR ITT appear to make the central tyrosine residue an even more favorable substrate for B cell-expressed protein tyrosine kinases such as Src and/or Syk, resulting in a more potent recruitment of Grb2.Furthermore, besides the ITT-like motif, there are two additional evolutionary conserved tyrosine residues in the cytoplasmic domain of FcµR, one of which, Y 366 , is embedded in a sequence (EYVSL) that resembles known phosphorylation and SH2 domain binding sites.Our results show that tyrosine residues other than that in the ITT-like motif are phosphorylated when the FcµR cytoplasmic domain is brought into proximity of activated BCRs.This raises the possibility that, for example, Y 366 serves as a phospho-acceptor and SH2 domain docking site to regulate additional intracellular signaling pathways.It remains to be elucidated how the Y 385 ITT-mediated signals and the aforementioned potential cleavage in the cytoplasmic tail of FcµR after ligation work. ",
"section_name": "Signaling Function of the Ig-Tail Tyrosine (ITT)-like Motif of FcµR",
"section_num": "4.2."
},
{
"section_content": "Using transductants stably expressing human or mouse FcµR, we observed different properties of the two orthologues in constitutively or transiently binding their IgM ligands, respectively [9].Via domain swapping analysis, this difference was directly attributed to the ligand-binding Ig-like domain rather than other parts of FcµR.The aa sequences of the Ig-like domain of human and mouse FcµRs were thus aligned with each other based on the secondary structure of human pIgR domain 1 (D1), determined by crystallography (PDB 5D4K) [37,38].Several sequence differences were found around the putative ligandbinding complementary determining regions (CDRs) that could account for the IgM binding difference between human and mouse receptors.In addition, two distinct stretches of 4-aa and 5-aa residues were also found in the presumed A ß strand and DE loop of FcµR, respectively [39].Based on this sequence comparison, we hypothesized that if non-conserved aa residues in human FcµR are replaced by the corresponding mouse residues, then the resultant human FcµR mutants may no longer constitutively bind IgM, similar to mouse FcµR.We thus replaced non-conserved aa residues of human FcµR with mouse equivalents and generated transductants stably expressing mutant or WT receptors.The eight different resultant FcµR mutant transductants were compared with WT transductants for their cell surface expression and IgM binding by flow cytometry using receptor-specific mAbs and IgM paraproteins as ligands.Since the details about receptor levels and IgM binding activity have been described elsewhere [39], the main findings are summarized below. Three sites of human FcµR, i.e., Asn 66 in the presumed CDR2, Lys 79 -Arg 83 in the DE loop, and Asn 109 in the CDR3, were responsible for its constitutive IgM binding since diminished IgM binding was observed for each replacement mutant without alteration of receptor expression levels.Notably, as previously presented [40], when all the above three sites of human residues were used to replace the mouse equivalents, the resultant mouse FcµR triple mutant (-66N, TPCLD78-82KQYPR, K108N) was found to constitutively bind IgM, such as human FcµR (Figure 5).The latter finding strongly supported that the aforementioned three sites were indeed critical for constitutive binding of IgM to human FcµR.Intriguingly, substitutions of Glu 41 and Met 42 in the CDR1, which is shorter than that of IgM-and IgA-binding receptors (pIgR and Fcα/µR), with the corresponding murine aa residues Gln and Leu, either alone or more prominently in combination, enhanced both the receptor levels and IgM binding activity.This finding was unexpected, because the mouse FcµR had limited or transient IgM binding activity as compared with human FcµR.While the molecular basis for this enhancement remains to be elucidated, this serendipitous finding may turn out to be informative when considering the design of therapeutic interventions targeting FcµR.A four-aa stretch of Lys 24 -Gly 27 in the predicted A ß strand of human FcµR appeared to be essential for maintenance of its receptor conformation on plasma membranes because of the reduction of both receptor expression and IgM binding potential when these were mutated.plasma membranes because of the reduction of both receptor expression and IgM binding potential when these were mutated. Collectively, the results from the site-directed mutational analyses suggest that the Asn residues at both positions 66 and 109 in the CDR2 and CDR3, respectively, and the stretch from Lys to Arg at positions of 79 to 83 in the DE loop of human FcµR, are responsible for its constitutive IgM-ligand binding activity.Collectively, the results from the site-directed mutational analyses suggest that the Asn residues at both positions 66 and 109 in the CDR2 and CDR3, respectively, and the stretch from Lys to Arg at positions of 79 to 83 in the DE loop of human FcµR, are responsible for its constitutive IgM-ligand binding activity. ",
"section_name": "Identification of Amino Acid Residues of FcµR Critical for IgM Binding",
"section_num": "4.3."
},
{
"section_content": "Recently, two groups of investigators, Junyu Xiao et al. (Peking University, Beijing) and Peter Rosenthal et al. (Francis Crick Institute, London), have independently released their pre-reviewed results of the structural basis for the interaction of FcµR with IgM, as determined by crystallography and cryo-electron microscopy (cryo-EM).The data are publicly available at the URLs provided at the end of the text.Although there were some differences in results (especially regarding stoichiometry) between these two studies, the essential findings were as follows.FcµR predominantly bound to the fourth constant domain (Cµ4) of IgM molecules.The key IgM-contacting aa residues of FcµR were: Arg 45 in the CDR1, Thr 57 and Thr 60 in the C' ß strand, Phe 67 and Lys 69 in the CDR2, and Thr 110 and Asp 111 in the CDR3.The FcµR-contacting aa residues of the IgM Cµ4 domain included Asn 465 , Leu 466 , Arg 467 , Glu 468 , and Glu 526 (Figure 6).Thus, none of the aa residues of FcµR predicted by our site-directed mutagenesis analysis were found to directly interact with IgM, but two of them, Asn 66 and Asn 109 , were immediately adjacent to the crystallographically identified contact residues Phe 67 and Thr 110 , respectively.Since, except for Thr 57 , six of the seven contact residues identified by crystallography are conserved in both human and mouse species, these residues were not selected by our site-directed mutagenesis strategy.The Lys 79 -Arg 83 stretch in the DE loop of FcµR was apparently not part of the IgMbinding interface, and hence, the finding of gain of constitutive IgM-binding by the mouse FcµR triple mutant remains puzzling and could be caused by an indirect conformational effect.Two Ig-like domains of FcµR were found to interact with both sides of the Cµ4 domain of the IgM monomer, suggesting a 2:1 stoichiometry for the FcµR/IgM monomer.Notably, both sides of the FcµR-binding Cµ4 domain of IgM BCR seemed to be accessible on the surface of B cells.In contrast, four FcµR molecules could bind the same side of the Cµ4 domain of an IgM pentamer in a way that once the first FcµR molecule had bound, then the remaining three receptors bound only to the same side in a certain cooperative manner, probably with the help of FcµR stalk regions, resulting in a 4:1 stoichiometry for the FcµR/IgM pentamer.The potential fifth Cµ4 domain was blocked by the J chain loop.According to the Rosenthal group, FcµR bound to both sides of the Cµ4 domain of the IgM pentamer (8:1 stoichiometry).The first FcµR binding was the site where pIgR D1 (or the secretory component) bound, probably due to its higher affinity.This suggested that FcµR could bind secretory IgM as well.Collectively, crystallographic and cryo-EM analyses reveal intricate mechanisms for the interaction of FcµR with monomeric and pentameric IgM molecules.The identification of aa residues of FcµR critical for IgM-ligand binding would facilitate screening of individuals with inborn errors of immunity, particularly FcµR deficiency. ",
"section_name": "Crystallographic Analysis of Human FcµR",
"section_num": "4.4."
},
{
"section_content": "",
"section_name": "Ex Vivo Functions of Human FcµR and Its Association with Diseases",
"section_num": "5."
},
{
"section_content": "FCMR deficiency has not yet been identified in humans but, if it exists, the clinical abnormalities might be much more complex and profound than in Fcmr-ablated mice, because additional cell types (i.e., T and NK cells) express the human FcµR.Thus, functional analyses of human FcµR rely on ex vivo experiments and several findings are noteworthy.(i) Co-ligation of FcµR and other receptors (e.g., Fas, CD2, BCR) on the same cell surface by agonistic IgM mAbs either inhibits Fas-mediated apoptosis or enhances BCR-or CD2mediated Ca 2+ mobilization, suggesting a dual (negative or positive) signaling ability [7,34,41,42].The cis engagement dominates the trans, probably due to locally high concentrations of IgM ligands [34].(ii) FcµR is highly expressed by chronic lymphocytic leukemia Collectively, crystallographic and cryo-EM analyses reveal intricate mechanisms for the interaction of FcµR with monomeric and pentameric IgM molecules.The identification of aa residues of FcµR critical for IgM-ligand binding would facilitate screening of individuals with inborn errors of immunity, particularly FcµR deficiency. ",
"section_name": "Ex Vivo Functions",
"section_num": "5.1."
},
{
"section_content": "FCMR deficiency has not yet been identified in humans but, if it exists, the clinical abnormalities might be much more complex and profound than in Fcmr-ablated mice, because additional cell types (i.e., T and NK cells) express the human FcµR.Thus, functional analyses of human FcµR rely on ex vivo experiments and several findings are noteworthy.(i) Co-ligation of FcµR and other receptors (e.g., Fas, CD2, BCR) on the same cell surface by agonistic IgM mAbs either inhibits Fas-mediated apoptosis or enhances BCRor CD2-mediated Ca 2+ mobilization, suggesting a dual (negative or positive) signaling ability [7,34,41,42].The cis engagement dominates the trans, probably due to locally high concentrations of IgM ligands [34].(ii) FcµR is highly expressed by chronic lymphocytic leukemia (CLL) B cells [7,33,[43][44][45] and, after IgM binding, it is rapidly internalized in lysosomes via an endocytic pathway [33].FcµR is not only present on the plasma membrane, but is also accumulated in large pools in the trans-Golgi network [33].(iii) FcµR on T and NK cells is dramatically down-modulated upon IL-2 stimulation in vitro, consistent with the finding that the surface levels of FcµR on freshly prepared effector memory T cells are much lower than on naïve T cells.Binding of IgM to FcµR on NK cells initiates intracellular signals but does not mediate NK cell cytotoxicity [42].(iv) FcµR-mediated IgM uptake by T cells enhances their surface expression of the T cell receptor and co-stimulatory molecules, thereby facilitating T cell activation, particularly when peptide antigen concentrations are low [46]. ",
"section_name": "Ex Vivo Functions",
"section_num": "5.1."
},
{
"section_content": "A variety of FcµR transcripts have been reported in the NCBI databases of both human and mouse species.However, only one splice variant other than its full length has been characterized at the protein level.It was originally identified in a phorbol myristate acetateactivated human pre-B cell line 697 by RT-PCR analysis with a set of primers corresponding to the translation initiation and termination sites of the human FcµR cDNA (NCBI gene accession: HM480394).It turned out that the alternatively spliced FCMR transcript resulted from the direct splicing of exon 4 (stalk-2) to exon 6 (cytoplasm-1), thereby skipping exon 5 (TM).This splicing event resulted in a frameshift in exon 6 and generated a novel 70-aa hydrophilic C-terminal tail, suggesting a soluble form of FcµR (solFcµR) (Figure 7A).The existence of such solFcµR in biological samples was confirmed when we assessed serum levels of FcµR in patients with CLL [45]. The association of the FcµR with CLL has long been suggested based on: (i) the ability of CLL cells to form rosettes with IgM-coated erythrocytes [47,48], (ii) IgM binding to CLL cells, as monitored by flow cytometry [49,50], and (iii) the expression of elevated levels of FCMR transcripts by microarray and RT-PCR analyses [43,44].We also examined the surface expression of FcµR on the B and T cells obtained from CLL patients using receptor-specific mAbs and flow cytometry [7,45] (Figure 7B).CLL B cells (CD5 + /CD19 + ) expressed much higher surface levels of FcµR than B cells from healthy donors.Such enhanced expression was more evident in better outcome or indolent CLL (i.e., Ig heavychain variable region (IGHV)-mutated, CD38 -, or early Rai-stage) than in poor outcome or aggressive CLL (i.e., IGHV-unmutated, CD38 + , or advanced Rai-stage).Notably, surface FcµR levels were also significantly elevated in non-CLL B cells (CD5 -/CD19 + ) and T cells (CD19 -/CD5 + ), especially in better outcome CLL patients, when compared with the corresponding populations in healthy donors [45]. script resulted from the direct splicing of exon 4 (stalk-2) to exon 6 (cytoplasm-1), thereby skipping exon 5 (TM).This splicing event resulted in a frameshift in exon 6 and generated a novel 70-aa hydrophilic C-terminal tail, suggesting a soluble form of FcµR (solFcµR) (Figure 7A).The existence of such solFcµR in biological samples was confirmed when we assessed serum levels of FcµR in patients with CLL [45].To determine whether CLL patients contain any soluble or extracellular (e.g., shed) form of FcµR in their sera, we performed a sandwich enzyme-linked immunosorbent assay (ELISA) using one anti-FcµR mAb (HM6 clone) as a capture antibody for serum FcµR and a biotin-labeled anti-FcµR mAb (HM14 clone) as a detection reagent (both HM6 and HM14 mAbs recognize a different epitope in the EC region of FcµR).Many CLL patients clearly exhibited elevated serum titers of FcµR, albeit with a wide range, as compared with healthy donors, except for one (see below).The titers were strongly correlated with the number of blood lymphocytes but not with CLL outcome indicators, including IGHV mutational statuses and Rai stages.Serum FcµR was resolved as a protein with an M r of ~40 kDa, thus being smaller than its membrane-bound counterpart of ~60 kDa (Figure 7B).By liquid chromatography tandem mass spectrometry analysis, the ~40 kDa serum FcµR was unequivocally defined as the aforementioned solFcµR.Notably, one of the tryptic peptides corresponded to the junction formed by splicing exon 4 to exon 6, ruling out the proteolytic cleavage of membrane-bound FcµR [45].The cellular source of the solFcµR in CLL patients appeared to be both CLL B and non-CLL B cells, but not T cells, as determined by RT-PCR and ELISA of their cell populations isolated by the fluorescence-activating cell sorter. Collectively, using receptor-specific mAbs, enhanced levels of both the ~60 kDa membrane-bound FcµR and the ~40 kDa solFcµR were demonstrated in patients with CLL compared with healthy donors.Enhanced surface FcµR expression was more evident in indolent CLL than aggressive CLL.Notably, the surface FcµR levels in both CD5 -non-CLL B cell and T cell populations were also significantly increased, especially in indolent CLL.The serum levels of solFcµR in CLL patients strongly correlated with their circulating lymphocyte counts but not with outcome indicators.The solFcµR was a product of an alternatively spliced FcµR transcript by both CD5 + CLL B and CD5 -non-CLL B cells. ",
"section_name": "Enhanced Levels of the Soluble Form of FcµR in Patients with Chronic Lymphocytic Leukemia",
"section_num": "5.2."
},
{
"section_content": "Among various leukemias and lymphomas, the enhanced expression of FcµR transcripts or proteins is predominantly observed in CLL [51].Unlike other B cell malignancies, BCRs on CLL cells are unique in that they can ligate each other via Ig heavy-chain CDR3 of one BCR with the framework region 2 of another BCR, regardless of their IGHV mutation statuses, thereby providing antigen-independent, cell-autonomous signaling [2,52] (Figure 7B).This antigen-independent homotypic BCR interaction on B CLL cells seems to affect the clinical course of disease as stronger affinity and longer half-life contacts are observed in indolent CLL, while weaker and more short-lived contacts are seen in aggressive CLL [53].This unique property of CLL B cells could account for the selective enhancement of FcµR expression in CLL, consistent with the finding that cross-linkage of BCR on normal blood B cells with antibodies enhanced the surface expression of FcµR [7].However, it was still unclear why surface FcµR levels were also elevated on non-CLL B and T cells, especially in indolent CLL patients.In this regard, CLL has been characterized by alterations of both adaptive and innate immune systems, including the roles of T cells, nurse-like cells, DCs, and bone marrow stromal cells in tumor surveillance and pathogenesis [54]. Another indication of the linkage between BCR ligation and enhanced FcµR expression was our observation of elevated levels of serum FcµR in one apparently 'healthy' individual who, however, developed high titers of anti-nuclear antibodies approximately two years after blood donation.We thus hypothesize that the production of solFcµR is a consequence of persistent BCR signaling.It is anticipated that the solFcµR levels may be elevated in individuals with other diseases characterized by chronic BCR stimulation, such as antibodymediated autoimmune disorders (Figure 7C).SolFcµR may modulate B cell function, either as a decoy receptor or by interacting with IgM BCR.In this regard, it is noteworthy that administration of a recombinant solFcµR variant (human FcµR EC/IgG Fc fusion protein lacking complement binding) into experimental allergic encephalomyelitis (EAE)susceptible mice ameliorates the myelin oligodendrocyte glycoprotein-induced EAE [17]. ",
"section_name": "Lessons from Studies on FcµR in CLL",
"section_num": "5.3."
},
{
"section_content": "Even though there are no reports describing the shedding of membrane FcµR by proteolytic cleavage, mAbs specifically recognizing the unique C-terminal portion of solFcµR as capturing reagents would be ideal in sandwich ELISA for assessments of the solFcµR in sera from patients with CLL or autoimmunity.This capturing strategy is clearly favorable over the FcµR EC-specific mAb (see above; Figure 7A).To develop such solFcµR-specific mAbs, we initially employed Ag8 plasmacytoma stably transduced by native solFcµR cDNA.However, because of its poor production, we modified the native solFcµR cDNA construct by introducing a consensus Kozak sequence directly 5 of the translation initiation site and inserting an 18 nucleotides coding six His residues tag before the termination codon.The resultant His-tagged solFcµR transductant produced 10-fold more solFcµR than the native solFcµR transductant.To our surprise, unlike the native solFcµR, the His-tagged solFcµR protein associated with extracellular membrane vesicles or exosomes, as well as with plasma membranes, rather than being in a free, secreted form [55]. Notably, the culture supernatants of His-tagged solFcµR transductants (even containing 10% fetal calf serum proteins) could be used for directly coating ELISA plates to immobilize solFcµR.This might imply that the exosome-attached form of His-tagged solFcµR proteins can efficiently adhere to polystyrene surfaces, as opposed to the free form of non-His-tagged solFcµR proteins.This unique property of His-tagged solFcµR was very convenient in the initial screening strategy by ELISA for hybridomas secreting solFcµR-specific mAbs without having to purify solFcµR proteins.In addition to this advantage, we also employed an in vivo differential immunization strategy using the solFcµR-His transductant as an immunogen and the membrane FcµR transductant as a tolerogen, resulting in generation of a new mouse IgG1κ mAb specific for human solFcµR [55].This mAb (HMD22 clone) will be used for assessments of a large cohort of patients with CLL or autoimmune disorders to verify the above hypothesis of solFcµR being a disease indicator as well as to identify the cells secreting the solFcµR. ",
"section_name": "Generation of mAbs Specific for solFcµR",
"section_num": "5.4."
},
{
"section_content": "Considering that IgM is the first phylogenetically emerging Ig isotype from jawed vertebrate onward, it seemed plausible to assume that FCMR orthologues are broadly distributed in various vertebrate species, as complement is.However, FcµRs appear to be selectively found in mammals [56].Another unexpected finding is that the expression of FcµR is restricted to lymphocytes: B, T, and NK cells in humans, and only B cells in mice.This implies that the lymphocyte-specific FcµR possesses distinct effector functions as compared with the FcRs for switched Ig isotypes that are expressed by various cell types, including phagocytes.From studies of Fcmr-deficient mice, FcµR has a regulatory function in B cell tolerance, as evidenced by their propensity to produce autoantibodies of both IgM and IgG isotypes (see the review in [13]).In this article, we have introduced our comments or frank opinions about two recent articles describing conflicting views-the regulation of anti-tumor activity by FcµR-bearing phagocytes infiltrating around tumors [19] and the reverse transcytosis of secretory IgM by FcµR expressed on the apical surface of M cells in Peyer's patches [27].The signal function of the Ig-tail tyrosine (ITT)-like motif seen in the FcµR cytoplasmic tail is now formally demonstrated by substitution of the IgG2a ITT.Unresolved aspects associated with our previous experiments of FcµR, i.e., its dual-signaling ability and its fast migration on SDS-PAGE after receptor ligation, were reiterated, with small progress.Recent crystallographic structural analyses of human FcµR by two different groups of investigators reveal seven key aa residues of FcµR directly contacting with the fourth constant domain of IgM as well as intricate mechanisms for the interaction of FcµR with the IgM monomer and pentamer.Differences in such aa residues as compared with our previous findings based on site-directed mutagenesis were discussed.The functional significance of the soluble form of FcµR, an alternative splice variant skipping the TM exon, has just begun to be explored in diseases characterized by persistent BCR stimulation, such as CLL and antibody-mediated autoimmune disorders.If this article ultimately facilitates research activity in the FcµR field by new investigators to resolve the many puzzles and unresolved issues as well as the functions, especially of human FcµR, the authors will be grateful and satisfied.We hope that this article will open new avenues of investigation. ",
"section_name": "Epilogue",
"section_num": "6."
}
] |
[
{
"section_content": "We thank the following investigators: David A. Hume, for his valuable comments for single-cell transcriptomic analysis of phagocytes; Brian Sutton and Rosaleen Calvert, for their collaboration in structural aspects of FcµR; Naganari Ohkura and Shimon Sakaguchi, for their epigenetic analysis of Treg cells; Peter D. Burrows, for his critical reading.We also thank the following colleagues: Takashi Kanaya, Hiroshi Ohno, Marie Burns, Eugene J. Becker, and Yoshiki Kubagawa for citing their unpublished observations, Hilmar Fünning for literature support, and Hilmar Frank for information technology support. ",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "Funding: This work was in part supported by the Leibniz Collaborative Excellence Grant CHROQ-K121/2018 (for F.M.), and by the Deutsche Forschungsgemeinschaft (DFG) through TRR130/Project 08 (for N.E.and J.W.). ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Author Contributions: Commentary work (Sections 2.1 and 2.2) was performed by C.M.S., P.M.A., P.K.J., F.M., A.R. and H.K.; ITT signaling (Section 4.2) by C.C., N.E.and J.W.; mutagenesis (Section 4.3) by C.M.S., K.A.-Q., R.T., A.R. and H.K.; solFcµR-specific mAbs (Section 5.3) by P.M.A., P.K.J., A.R., F.M. and H.K.; aspects in other sections are indicated in the cited references.J.W., N.E.and H.K. wrote the manuscript.All authors have read and agreed to the published version of the manuscript. The authors have no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "Author Contributions: Commentary work (Sections 2.1 and 2.2) was performed by C.M.S., P.M.A., P.K.J., F.M., A.R. and H.K.; ITT signaling (Section 4.2) by C.C., N.E.and J.W.; mutagenesis (Section 4.3) by C.M.S., K.A.-Q., R.T., A.R. and H.K.; solFcµR-specific mAbs (Section 5.3) by P.M.A., P.K.J., A.R., F.M. and H.K.; aspects in other sections are indicated in the cited references.J.W., N.E.and H.K. wrote the manuscript.All authors have read and agreed to the published version of the manuscript. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors have no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.3389/fphar.2021.628209
|
Effect and Mechanism of Catalpol on Remyelination via Regulation of the NOTCH1 Signaling Pathway
|
<jats:p>Promoting the differentiation of oligodendrocyte precursor cells (OPCs) is important for fostering remyelination in multiple sclerosis. Catalpol has the potential to promote remyelination and exert neuroprotective effects, but its specific mechanism is still unclear. Recent studies have shown that the NOTCH1 signaling pathway is involved in mediating OPC proliferation and differentiation. In this study, we elucidated that catalpol promoted OPC differentiation<jats:italic>in vivo</jats:italic>and vitro and explored the regulatory role of catalpol in specific biomolecular processes. Following catalpol administration, better and faster recovery of body weight and motor balance was observed in mice with cuprizone (CPZ)-induced demyelination. Luxol fast blue staining (LFB) and transmission electron microscopy (TEM) showed that catalpol increased the myelinated area and improved myelin ultrastructure in the corpus callosum in demyelinated mice. In addition, catalpol enhanced the expression of CNPase and MBP, indicating that it increased OPC differentiation. Additionally, catalpol downregulated the expression of NOTCH1 signaling pathway-related molecules, such as JAGGED1, NOTCH1, NICD1, RBPJ, HES5, and HES1. We further demonstrated that<jats:italic>in vitro</jats:italic>, catalpol enhanced the differentiation of OPCs into OLs and inhibited NOTCH1 signaling pathway activity. Our data suggested that catalpol may promote OPC differentiation and remyelination through modulation of the NOTCH1 pathway. This study provides new insight into the mechanism of action of catalpol in the treatment of multiple sclerosis.</jats:p>
|
[
{
"section_content": "Multiple sclerosis (MS), a demyelinating disease, affects the central nervous system (CNS), especially the white matter, and is characterized by immune cell infiltration, demyelination, oligodendrocyte (OL) loss and axonal destruction (McQualter and Bernard, 2007;Jurasic et al., 2019).Demyelination caused by immune inflammation results in severe neurological dysfunction in patients with MS (Stanojlovic et al., 2016).Promoting remyelination is important for the restoration of neurological function in MS.During the development of MS, lesions contain enough oligodendrocyte precursor cells (OPCs), which differentiate into myelinating OLs, to form a new myelin sheath around the injured axon.However, this process fails due to interference by various factors.A previous study revealed that activation of the NOTCH1 signaling pathway is one of these factors.The NOTCH1 signaling pathway affects the development and progression of the disease in the CNS by regulating the proliferation, differentiation and apoptosis of stem cells.NOTCH1 binds to the ligand JAGGED1to activate downstream signaling pathways and then directly converts extracellular information into changes in nuclear gene expression (Zhang et al., 2009;Paganin and Ferrando, 2011), which may hinder OPC differentiation and remyelination. Doctors often use glucocorticoids and plasma exchange to alleviate symptoms, shorten the course of the disease, reduce the degree of disability and prevent complications in the acute phase of MS.In the remission phase, teriflunomide, interferon, fingolimod and other disease-modifying therapies (DMTs) are used to control disease progression and reduce recurrence.However, these therapies cannot prevent CNS neurodegeneration (Faissner et al., 2019).Currently, there are no safe and effective strategies to promote remyelination to improve nerve function (Plemel et al., 2017;Katsara and Apostolopoulos, 2018). Catalpol, also known as catalpinoside, is the main active ingredient of the traditional Chinese herbal medicine Rehmannia glutinosa (Gaertn.)DC.. Catalpol has anti-inflammatory and antioxidant properties and exerts neuroprotective effects, improving neurocognitive function (Xia et al., 2017).Administration of 10 mg/kg or 20 mg/kg catalpol for 14 days produces significant antidepressant effects in a mouse model of depression through the serotonin pathway (Wang et al., 2014).The antidepressant effects of catalpol may be related to repair of the hypothalamic-pituitaryadrenal (HPA) axis and increased expression of brain-derived neurotrophic factor (BDNF) (Wang et al., 2015).Catalpol can also protect forebrain neurons from neurodegeneration and enhance memory by increasing BDNF expression (Liu et al., 2006;Wang et al., 2009;Wan et al., 2013).In an experimental model of Parkinson's disease, catalpol increases the concentration of striatal dopamine and the level of glial cell-derived neurotrophic factor (GDNF) (Xu et al., 2010), thereby exerting its neuroprotective functions. We have found that catalpol can promote remyelination in experimental autoimmune encephalomyelitis (EAE) mice by upregulating the expression of the transcription factors OLIG1 and OLIG2, as well as increasing the proliferation, migration and differentiation of OPCs in vitro (Yuan et al., 2015;Yang et al., 2017).However, the mechanism is unclear.Therefore, in this study, we will use a mouse model of demyelination induced by cuprizone (CPZ) and OPCs in vitro to explore whether catalpol promotes remyelination by regulating the NOTCH1 signaling pathway. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "",
"section_name": "Animals",
"section_num": null
},
{
"section_content": "Special feed containing 0.2% CPZ (Sigma, United States) produced by Beijing Keao Xieli Feed Co., Ltd. [Beijing Feed Certification (2014) 06054] was used for this experiment after disinfection by cobalt-60 irradiation.For in vivo studies, catalpol was purchased from Nanjing Dilge Pharmaceutical Technology Co., Ltd. and used in doses of 20 mg/kg, 40 mg/kg and 80 mg/kg, and its purity was ≥85%.For in vitro studies, the standard substance of catalpol was provided by Target Molecule Corp (T2780) and used in the concentrations of 0-100 μM, and its purity was ≥ 99%. ",
"section_name": "Drugs",
"section_num": null
},
{
"section_content": "Sixty mice were randomly divided into five groups: the normal control (NC) group (n 15), the model (MO) group (n 15), and the 20 mg/kg, 40 mg/kg and 80 mg/kg catalpol groups (n 10/ group).The mice in the NC group were given normal feed, and the mice in the other five groups were given feed containing 0.2% CPZ.After 5 weeks, the levels of relevant indicators were assessed in five mice from each of the NC group and the MO group.From the 6 th week, the remaining mice were provided normal feed.The catalpol groups were administered the corresponding dose of catalpol daily by gavage, whereas the NC and MO groups were given the same volume of normal saline.Biological materials were collected after 8 weeks (Figure 1). ",
"section_name": "Establishment and Treatment of the CPZ-Induced Demyelination Mouse Model",
"section_num": null
},
{
"section_content": "From the start of modeling, the body weight of each mouse was measured twice a week.For three days before modeling, the mice were subjected to balance training on a rotating device twice a day.After the mice had been adapted to the device, their motor abilities were tested twice a week.The rotation speed was slowly increased from 5 rpm to 40 rpm over 3 min.When a mouse fell off or held onto the rotating rod and for two or more rotations, the experiment was ended.The time spent on the rod by the mice was recorded to monitor changes in coordination and balance (Fan et al., 2018). ",
"section_name": "Body Weight Measurement and the Rotarod Test",
"section_num": null
},
{
"section_content": "The mice were anesthetized with 4% chloral hydrate and perfused with 40 g/L paraformaldehyde for 30 min, and their tissues were embedded with paraffin.Five-micron -thick coronal slices of the brain located in the corpus callosum were prepared.After being washed with phosphate-buffered saline (PBS), the brain slices were baked at 60 °C for 2 h, placed in 1:1 ethanol and chloroform for 4 h, and then transferred to 95% ethanol.After dehydration, the sections were placed in LFB solution overnight, rinsed, and then subjected to color development with 0.05% lithium carbonate and 70% alcohol.After being rinsed, the sections were counterstained with cresyl violet for 5 min, rinsed with water, dehydrated, removed, and sealed for observation. ",
"section_name": "Luxol Fast Blue Staining",
"section_num": null
},
{
"section_content": "Newborn Sprague-Dawley (SD) rats were provided by Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. [SCXK (Beijing) 2016-0006].OPCs were isolated and purified by a method involving B104-conditioned medium.Mixed glial cells were isolated from the cortices of newborn rats.After being cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) for 3 days, the mixed glial cells were cultured with modified OPC growth medium (mOGM) containing 15% B104-conditioned medium.The OPCs were isolated and purified by a chemical-based separation procedure when they had proliferated enough. ",
"section_name": "Primary OPC Culture",
"section_num": null
},
{
"section_content": "One hundred microliters of a single-cell suspension containing OPCs (1.5 × 10 4 ) was seeded in PPL-coated 96-well plates for 24 h and then treated with catalpol (0, 1, 2.5, 5, 10, 20, 40, 80, or 100 μM) for 24 h, 48 h or 72 h.Then, cell viability was assessed by a CCK-8 kit according to the manufacturer's instructions.The optical density (OD) values at 450 nm were measured with a microplate reader and normalized to those of the control groups. ",
"section_name": "Cell Counting Kit (CCK)-8 Assay",
"section_num": null
},
{
"section_content": "After perfusion, the corpus callosum tissues of the mice were separated on ice, cut into approximately 1 × 1 × 3 mm 3 pieces, placed in 2.5% glutaraldehyde for 2 h, and then rinsed with 0.1 M PB three times.Then the corpus callosum tissues were fixed with 1% osmium acid, dehydrated in alcohol, soaked for 20 min, embedded in embedding agent, subjected to melt impregnation and treated with pure embedding agent.The embedded samples were sliced with an ultrathin microtome and then stained and coverslipped.The ultrastructure of the myelin sheath was observed with an electron microscope (JEM-2100, JEOL, Tokyo, Japan).We randomly selected at least 80 axons from each group, used professional image analysis software (Image-Pro Plus, IPP) to measure the diameter of the myelin sheath and axon in each field of view, and calculated the G-ratio (axon/axon diameter + myelin sheath diameter) to evaluate demyelination. ",
"section_name": "Transmission Electron Microscopy",
"section_num": null
},
{
"section_content": "The mouse brain slices were dewaxed, hydrated, incubated in citric acid for 20 min for antigen repair, cooled to room temperature (RT), and blocked with 10% goat serum at 37 °C for 60 min.Goat anti-OLIG2 (1:100; R&D, MN, United States), mouse anti-CNPase (1:200, Abcam, Cambridge, United Kingdom), rabbit anti-MBP (1: 200, Abcam, Cambridge, United Kingdom), and rabbit anti-GFAP (1:400, Abcam, Cambridge, United Kingdom) primary antibodies were added dropwise, and the slices were incubated at 4 °C for 48 h.After the slices were rewarmed at 37 °C for 60 min, they were incubated with corresponding fluorescently labeled IgG antibodies (Alexa Fluor 488-conjugated donkey anti-rabbit or mouse IgG [1: 200], Cy3-labeled donkey anti-goat IgG [1: 200], or Alexa Fluor 488-conjugated goat anti-rabbit IgG [1: 200]).Then, the slices were sealed with DAPI solution.Representative images were obtained with the Pannoramic SCAN digital slice scanner and analysis software (3DHISTECH, Hungary), and ImageJ was used to determine the expression of the abovementioned proteins in the selected area for further data analysis. A total of 500 μl of a single-cell suspension containing OPCs (4.5 × 10 4 ) was plated in 24-well plates with coated glass coverslips and incubated for 12 h.Then, catalpol (0, 1, 2.5, 5, 10, 20, 40, 80, or 100 μM) was added.After 24 h, 48 h or 72 , the OPCs on coverslips were fixed with 4% paraformaldehyde for 30 min at RT and treated with 0.5% Triton for 10 min.After being rinsed with PBS, the coverslips were blocked with 5% bovine serum albumin (BSA) for 1 h and then incubated with a rabbit anti-MBP antibody (1:200, Abcam, Cambridge, United Kingdom) at 4 °C overnight.Then, the cells were washed with PBS and incubated with Alexa Fluor 488conjugated donkey anti-rabbit IgG (1:200) at RT for 1 h.The cell nuclei were stained with DAPI for 5 min.Immunoreactivity was observed using a fluorescence microscope.All images were analyzed with ImageJ software. ",
"section_name": "Immunofluorescence",
"section_num": null
},
{
"section_content": "OPCs (9 × 10 5 cells/well in 6-well plates, treated with 40 μM catalpol according to the method used for immunofluorescence) and mouse brain tissues (containing the corpus callosum, cortex and hippocampus, 30-50 µg) were lysed with RIPA lysis buffer.The protein concentrations were measured using the Bicinchoninic Acid (BCA) Protein Assay Kit and normalized.The proteins were separated by SDS-PAGE and transferred onto PVDF membranes.After being blocked with 5% skim milk for 1 h, the membranes were incubated with the following primary antibodies overnight at 4 °C: rabbit anti-JAGGED1 (1:1,000, CST, 2620), rat anti-NOTCH1 (1:1,000, CST, MA, United States), rabbit anti-NICD1 (1:1,000, CST, MA, United States), rabbit anti-RBPJ (1:1,000, CST, MA, United States), rabbit anti-HES1 (1:1,000, CST, MA, United States), rabbit anti-HES5 (1:1,000, CST, MA, United States), mouse anti-ACTB (1:20,000, Gene Tex, CA, United States), and mouse anti-TUBB (1:20,000, Proteintech, Chicago, United States).After being washed, the membranes were incubated with corresponding IgG antibodies (1:10,000) at RT for 1 h.The proteins were exposed by a gel chemiluminescence imaging analysis system using enhanced chemiluminescence (ECL) reagent.ImageJ software was used to process the images, determine the grayscale values of the bands, and semiquantitatively analyze the relative expression of each protein. ",
"section_name": "Western Blot Analysis",
"section_num": null
},
{
"section_content": "Total RNA was extracted from 50 μg samples with the One-Step qRT-PCR kit (Toyobo, Osaka, Japan), and then the concentration was measured.RT-PCR mixtures were prepared, and qRT-PCR was performed by the CFX96 TM Real-Time PCR instrument to confirm the mRNA expression levels of the target genes.Actb was used as an internal reference for RNA.mRNA expression levels were quantified using the Bio-Rad CFX Real-Time system and analyzed using CFX management software v2.0 (Bio-Rad, Hercules, CA) and the 2 -△△ Ct method. The sequences of the primers used in this study were as follows: ",
"section_name": "Quantitative RT-PCR",
"section_num": null
},
{
"section_content": "All data are presented as the mean ± SEM.Statistical analyses were performed by one-way analysis of variance and Tukey's HSD multiple comparison test using GraphPad Prism 7.0 software.For all statistical tests, p-values < 0.05 were considered statistically significant. ",
"section_name": "Statistical Analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "The body weights of the mice in the NC group increased over time.The weights of the mice in the MO (5 weeks) group gradually decreased to the lowest by the end of the second week and then slowly increased.There was a statistically significant difference in body weight between the NC and MO (5 weeks) groups (p < 0.05, Figure 2A).The results of the rotarod test showed that the time spent on the rod by the mice in the NC group remained basically the same over time.However, from the 3rd week, the time spent on the rod by the mice in the MO (5 weeks) group decreased significantly (p < 0.05, Figure 2B). LFB staining indicated that in the mice in the NC group, the myelin sheaths were tight and properly arranged and that there was a large amount of staining; however, in the MO (5 weeks) group, myelin staining in the corpus callosum was notably reduced and sparse, or even absent (p < 0.05, Figure 2C).According to the results of TEM, in the NC group, the myelin sheaths were clear and dense and no demyelination or axonal atrophy was observed, whereas ",
"section_name": "Successful Establishment of the CPZ-Induced Demyelination Model",
"section_num": null
},
{
"section_content": "After the mice were given normal feed beginning in the 6 th week, the body weights of the mice in the NC group continued to increased, and those of the mice in the MO group recovered slightly (p < 0.05).The body weights of the mice in the 40 mg/kg and 80 mg/kg catalpol groups recovered quickly.From the 7 th week, there were significant differences in body weight between the catalpol groups and the MO group (p < 0.05, Figure 3A).The rotarod test results showed from the 6 th week, the time spent on the rod by the mice in the NC group remained relatively stable, whereas that spent by the mice in the MO group improved but was still significantly shorter than that spent by the mice in the NC group (p < 0.05).The time spent on the rod by the mice in the 20 mg/kg catalpol group was significantly longer than that spent by the mice in the MO group from the 7 th week (p < 0.05), while the time spent on the rod by the mice in the 40 mg/kg and 80 mg/kg catalpol groups was significantly longer than that spent by the mice in the MO group beginning at six and a half weeks (p < 0.05, Figure 3B). ",
"section_name": "Catalpol Increased the Body Weights and Improved the Motor Functions of Demyelinated Mice",
"section_num": null
},
{
"section_content": "At the 8 th week, partial remyelination was observed in the corpus callosum in mice in the MO group.The loss of LFB staining of myelin and myelin lamination was slightly delayed in the MO group, but a significant difference compared with the NC group (p < 0.001) was still observed.The stained myelin sheaths were significantly denser and better arranged (Figure 4A), the demyelinated area was significantly smaller (Figure 4B) in the catalpol administration groups than in the MO group (p < 0.05).The three catalpol groups exhibited loosening of the lamellar structure of myelin to varying degrees and decreases in ring density.However, these changes were less severe in the three catalpol groups than in the MO group (Figure 4C).The G-ratios were significantly lower in the catalpol groups than in the MO group (p < 0.05, p < 0.001), with the 40 mg/kg and 80 mg/kg catalpol groups exhibiting more significant changes (p < 0.05, p < 0.001, Figure 4D). ",
"section_name": "The Effect of Different Concentrations of Catalpol in Reducing Myelination in CPZ-Induced Demyelination Mice",
"section_num": null
},
{
"section_content": "CNPase and MBP are markers of OL differentiation and maturity, respectively.OLIG2, which is essential for cell fate choices, is a transcription factor in OLs (Gouvea-Junqueira et al., 2020).The expression levels of CNPase/OLIG2 and MBP/OLIG2 in the brains of mice in each group were detected by immunofluorescence to evaluate the differentiation and maturation of OPCs.The experimental results indicated that there was no difference in the expression of OLIG2 in the corpus callosum between groups (Figure 5B).This finding further indicates that insufficient differentiation of OPCs, not a lack of OPCs, causes the failure of remyelination in MS.Furthermore, the expression levels of CNPase/OLIG2 in the mice in the MO group were decreased compared to those in the NC group (Figure 5A, p < 0.001); CNPase/OLIG2 levels in the catalpol treatment groups were increased to a certain extent.Catalpol had more robust effects at 40 mg/kg and 80 m/kg than at 20 mg/kg (p < 0.01).The expression of CNPase protein was also measured by WB analysis (Figure 5D), and similar results were obtained.The MBP measurement results were basically consistent with those of CNPase.There was no difference in the expression of OLIG2 between groups (Figure 6B), but there were obviously fewer MBP + /OLIG2 + cells in the MO group than in the NC group and the catalpol groups (Figure 6A, p < 0.001).Analysis of MBP protein expression in the brain further confirmed the effect of catalpol on promoting the differentiation of OPCs (Figure 6D). ",
"section_name": "Catalpol Upregulated the Expression of CNPase and MBP in the Corpus Callosum in Demyelinated Mice",
"section_num": null
},
{
"section_content": "GFAP is a marker of astrocytes.Studies have found that astrocytes participate in the first line of defense against the early stages of immune inflammation in MS (Farina et al., 2007;Brosnan and Raine, 2013;Ponath et al., 2017) and have neuroprotective effects (Colombo and Farina, 2016).However, the activation of astrocytes during the remyelination stage is harmful (Mayo et al., 2012).Reactive astrocytes form an astroglial scar with recruited chondroitin sulfate proteoglycans, hyaluronic acid and other molecules, the levels of which are upregulated, affecting remyelination (Liddelow et al., 2017).On the other hand, a large number of astrocytes produce platelet-derived growth factor α and fibroblast growth factor 2, which inhibit the differentiation of OPCs.In this study, we found that the expression of GFAP was elevated in the MO group compared with the NC group (p < 0.001).At the three tested doses, of catalpol decreased GFAP expression in the brain to varying degrees (p < 0.01, Figure 7). ",
"section_name": "Catalpol Downregulated the Expression of GFAP in the Brains of Demyelinated Mice",
"section_num": null
},
{
"section_content": "The NOTCH1 signaling pathway is active in the nervous system throughout life and plays a role in maintaining the steady state of stem or progenitor cells in the developing CNS (Chitnis et al., 1995;Wettstein et al., 1997).NOTCH1 signaling alters the proliferation and differentiation of differentiated cells through cell-to-cell communication.The activation of NOTCH1 ligands and downstream target genes of the Hes family blocks the differentiation of OPCs, causing the failure of myelination by OLs.These findings indicate that the NOTCH1 pathway may be primarily responsible for the failure of remyelination in MS The data are expressed as mean ± SEM (n 3/each group), compared with NC group, ***p < 0.001; compared with MO group, # p < 0.05, ## p < 0.01, ### p < 0.001.(Mathieu et al., 2019).Therefore, in this experiment, we mainly assessed the levels of several indicators related to the NOTCH1 signaling pathway: the ligand JAGGED1; the transmembrane receptor NOTCH1 (Fortini, 2009;Kovall et al., 2017); Notch intracellular domain (NICD), which is released by γ-secretase; recombination signal binding protein for immunoglobulin kappa J region (RBPJ), and the downstream transcription factors HES1 and HES5 (Tamura et al., 1995). We observed that the protein and gene levels of NOTCH1(Notch1), JAGGED1 (Jag1), NICD1, RBPJ (Rbpj), HES1(Hes1), and HES5 (Hes5) were significantly increased in mice in the MO group compared with mice in the NC group 40 mg/kg or 80 mg/kg catalpol than those of mice in the MO group (p < 0.05, p < 0.01, Figure 8C); all doses of catalpol downregulated the expression of RBPJ (Rbpj) to a certain extent, with 80 mg/kg catalpol having the most significant effect (p < 0.05, Figure 8D); the protein and gene levels of HES1 (Hes1) in the brain were obviously decreased in the three catalpol treatment groups compared to the MO group, with 80 mg/kg catalpol having the most significant effect (p < 0.05, Figure 8E); and the downward trend in HES5 (Hes5) expression was similar to that of HES1 (Hes1) (Figure 8F). ",
"section_name": "Effects of Catalpol on NOTCH1 Signaling Pathway-Related Proteins and Genes in the Brains of Demyelinated Mice",
"section_num": null
},
{
"section_content": "We successfully isolated OPCs from the brains of suckling rats and cultured them in vitro.We used PDGFRα, a marker of OPCs, to identify the cultured cells.Next, OPCs were cultured in differentiation medium, and the cells at different stages from OPCs to OLs were labeled with markers at different stages to prove that the cultured OPCs had strong differentiation ability (Figure 9). ",
"section_name": "Cultivation of OPCs and OLs",
"section_num": null
},
{
"section_content": "To study the effect of catalpol on the activity of OPCs, cells were treated with catalpol (1, 2.5, 5, 10, 20, 40, 80, or 100 μM) for 24, 48 or 72 , and cell viability was measured by the CCK-8 assay.Catalpol increased the viability of OPCs to different extents (Figure 10A). ",
"section_name": "Catalpol Increased the Viability of Primary OPCs",
"section_num": null
},
{
"section_content": "MBP is a marker of mature OLs.Catalpol significantly increased the number of MBP + cells.The results revealed that treatment with 0-100 μM catalpol for 24-72 h promoted the differentiation of OPCs into OLs (Figure 10B). According to the findings related to the viability of and MBP expression in OPCs, catalpol had the best effect at a concentration of 40 μM for 48 h (Figure 10C).Therefore, we chose treatment with 40 μM catalpol for 48 h for further experiments in vitro. ",
"section_name": "Catalpol Promoted the Formation of Mature OLs In Vitro",
"section_num": null
},
{
"section_content": "To investigate whether the NOTCH1 signaling pathway can influence the differentiation of OPCs, OPCs were seeded in 6well plates and treated with 40 μM catalpol in the absence or presence of the NOTCH1 signaling pathway agonist JAGGED1 polypeptide for 48 h.Treatment with 5 μM JAGGED1 for 24 h blocked the formation of MBP + OLs, and the addition of catalpol reversed this effect (Figure 11F).The protein levels of NICD1, RBPJ, HES1 and HES5 effectively increased in OPCs in the JAGGED1 group (JAG1).The expression of the above indicators was significantly lower in cells treated with catalpol in the presence of JAGGED1 than in cells in JAG1 group (Figures 11A-E).Taken together, our findings indicate that the stimulation of OL formation by catalpol is in part due to the suppression of the NOTCH1 signaling pathway. ",
"section_name": "Catalpol Promoted the Differentiation of OPCs In Vitro via the NOTCH1 Signaling Pathway",
"section_num": null
},
{
"section_content": "",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "CPZ is a copper ion-chelating agent that targets many metalloenzymes (such as ceruloplasmin), impairs the activity of copper-dependent cytochrome oxidase, and reduces oxidative phosphorylation, leading to degenerative changes in OLs (Cammer, 1999).These changes result in the apoptosis of mature OLS, causing extensive demyelination of the corpus callosum, internal capsule, thalamus and other white matter bundles (Blakemore, 1972;Clarner et al., 2015).However, after The ratio of quantitative analysis of the fluorescent expression of MBP in each group to the untreated (Catalpol, 0 μM) group.The data are expressed as the mean ± SEM of three independent experiments, compared with catalpol 0 μM for 24 h group, *p < 0.05, **p < 0.01, ***p < 0.001; compared with catalpol 0 μM for 48 h group, # p < 0.05, ## p < 0.01, ### p < 0.001; compared with catalpol 0 μM for 72 h group, △ p < 0.05, △△ p < 0.001. CPZ administration is stopped, the myelin protein is reexpressed.Therefore, although pathological changes in autoimmunity observed in MS can be stimulated by the EAE model, the demyelination model induced by the addition of CPZ to the diet, which also mimics the important histological features of demyelinating diseases (van der Star et al., 2012), is the ideal model for researching myelination in MS (Gudi et al., 2014).The exact dosage of CPZ has been determined by several studies.The most common protocol involves feeding 6-to 8-week-old mice with 0.2% CPZ for 5-6 weeks (Vega-Riquer et al., 2019).When 0.2%-0.6%CPZ is mixed with standard rodent food, a significant decrease in myelin protein is observed (Carlton, 1967).Studies have shown that increasing the dose of CPZ from 0.2% to 0.3% can significantly increase the degree of demyelination (Lindner et al., 2008) but increases the mortality rate of mice by more than 5%-10% (Torkildsen et al., 2008).Therefore, 0.2% CPZ is the most suitable concentration because it can cause extensive demyelination and fewer side effects (Liñares et al., 2006;Hesse et al., 2010;Skripuletz et al., 2011). This experiment mainly studied the effect of catalpol on promoting myelination and neuroprotection, so the CPZinduced demyelination model was selected.The results showed that after mice were fed a special diet containing 0.2% CPZ for 5 weeks, the mice exhibited a substantial decrease in weight, deficits in motor ability, and a reduction in LFB staining of myelin in the corpus callosum.The lamellar structure of the myelin sheath was obviously loose and separated, and the G-ratio value was increased.We found that after the demyelinated mice were fed a normal diet for 3 weeks beginning during the 6 th week, the weights and motor abilities of the mice slowly recovered, LFB staining of myelin in the corpus callosum increased, and the loosening of the myelin layers decreased, indicating that some myelin sheaths had regenerated. ",
"section_name": "Establishment of the CPZ-Induced Demyelination Model",
"section_num": null
},
{
"section_content": "When demyelinating injury occurs in MS, the damaged myelin fragments activate the immune inflammatory response and recruit a large amount of infiltrating immune inflammatory cells.Excessive inflammatory responses cause neuronal damage and severe neurological dysfunction in MS patients (Paganin and Ferrando, 2011).Catalpol exerts a strong neuroprotective effect and improves neurocognitive function (Xia et al., 2017) by reducing the level of proinflammatory cytokines and reducing oxidative stress in the nervous system, thereby slowing chronic inflammation and neurodegeneration (Wang et al., 2019).Previous studies have found that catalpol decreases immune inflammation and improves nerve damage in EAE mice (Yang et al., 2017).The results of this experiment revealed that at different doses, catalpol slowed the loss of myelin and the loosening of the myelin structure in the corpus callosum in mice, improved the body weight of the mice and increased the time spent on the rod by the mice in the rotarod test, showing that catalpol exerts good neuroprotective effects in mice with CPZinduced demyelination.Catalpol had a better effect at 40 mg/kg and 80 mg/kg than at 20 mg/kg. ",
"section_name": "Neuroprotective Effects of Catalpol",
"section_num": null
},
{
"section_content": "When demyelination occurs, axonal conduction is blocked (Waxman, 1977), and this effect is closely related to the functional defects observed after inflammatory demyelination in MS patients (Smith and McDonald, 1999;Jenkins et al., 2010).Therefore, prevention of demyelination and promotion of remyelination are the basic neuroprotective strategies for MS (Plemel et al., 2017;Villoslada and Steinman, 2020).In the mouse model of CPZ-induced demyelination, the regeneration of myelin in the injured area is closely related to functional recovery (Mozafari et al., 2010), which is extensive and rapid.The proliferation and recruitment of OPCs after injury to the mature CNS is very effective.Neural stem cells or progenitor cells from the subependymal zone rapidly produce OPCs that contribute to myelination (Xing et al., 2014), and OLs newly formed by OPCs undergo remyelination (Crawford et al., 2016).Studies have shown that the change between the average density of OPCs in chronically damaged sites in MS and normal sites is not obvious, which indicates that the number of OPCs may not be the limiting factor for remyelination in chronic injury, proving the importance of the differentiation of OPCs during remyelination (Hughes et al., 2013). During the process of myelination in MS, OLIG2 acts as a cell transcription factor throughout the differentiation of OL lineage cells and can simultaneously label OPCs and OLs.The experimental results showed that the expression of OLIG2 in the corpus callosum increased slightly in all demyelinated mice, but there was no significant difference between the groups, indicating that OPCs in the demyelinated area were not lacking.CNPase is expressed in the middle stage of differentiation, while MBP is a marker of mature OLs.Early in vitro studies have found that catalpol increases the expression of OLIG1 in isolated OPCs and promotes the differentiation and maturation of OPCs (Yuan et al., 2015).This experiment revealed that catalpol increased the number of CNPase + / OLIG2 + and MBP + /OLIG2 + cells in the demyelination site and that 40 μM catalpol promoted the differentiation of OPCs in vitro.The results provide an objective basis for catalpol to promote myelination. ",
"section_name": "Catalpol Promoted Remyelination",
"section_num": null
},
{
"section_content": "In MS, remyelination is always insufficient.The failure of myelination appears to be the result of a variety of pathological processes that interfere with the maturation of OPCs.The activation of the NOTCH1 signaling pathway is one of these processes (Mathieu et al., 2019).It has been reported that the NOTCH1 signaling axis is activated in response to TGF-β in the brains of MS patients and in cocultured astrocytes and OPCs in vitro.As in normal development, JAGGED1 is expressed in astrocytes and neurons in chronic demyelinating lesions, and the receptor NOTCH1 on OPCs combines with activated JAGGED1 and undergoes a conformational change.Subsequently, ADAM metalloprotease mediates the first protein cleavage, producing an intermediate protein hydrolysate called Notch EXtracellular Truncation (NEXT).NEXT is the substrate for the γ-secretase complex, which releases NICD.NICD passes through the cytoplasm from the inner membrane of the plasma membrane to the nucleus.In the cell nucleus, after NICD combines with RBPJ, RBPJ is converted into a transcription activator that recruits acetyltransferase p300 and activates the downstream transcription factors HES1 and HES5 (Tamura et al., 1995).The transcription factors HES1 and HES5 inhibit the maturation of OPCs and maintain their differentiation status.Therefore, blocking NOTCH1 signaling may enhance remyelination. In EAE mice, the γ-secretase inhibitor MW167 can effectively inhibit the NOTCH1 pathway and promote remyelination (Jurynczyk et al., 2008).In mice with focal demyelination caused by injection of lysophosphatidylcholine, specific knockout of NOTCH1 in OPCs can significantly improve myelin repair (Zhang et al., 2009).NOTCH1 siRNA can obviously promote OPC differentiation and promote remyelination to improve the symptoms of nerve injury in CPZ mice (Fan et al., 2018).The above studies revealed that inhibiting the NOTCH1 signaling pathway can improve the disease pathology of MS in animal models and enhance the regeneration of the myelin sheath.Other studies have found that when the expression of jagged1 in astrocytes is induced by TGFbeta1, OL differentiation increases (Zhang et al., 2010).Adding 10 µM DAPT (a γ-secretase inhibitor) to an OPC and astrocyte coculture system for 6 h can significantly inhibit the expression of NICD and promote cell differentiation (Wang et al., 2017).This study showed that in vivo, catalpol significantly promoted remyelination in CPZ-treated mice and decreased the expression of the NOTCH1 signaling pathway proteins JAGGED1, NOTCH1, NICD1, and RBPJ and the downstream target genes Hes1 and Hes5 and 80 mg/kg catalpol had the best therapeutic effect.Multiple studies have proven that inhibition of the NOTCH1 signaling pathway can promote the differentiation and maturation of OPCs in vivo and in vitro and promote the remyelination of demyelinated mice.Furthermore, this study revealed that the NOTCH1 pathway is strongly activated in OPCs upon treatment with 5 μM JAGGED1 polypeptide for 24 h.The number of MBP + cells as also significantly reduced.Treatment with 40 μM catalpol for 48 h greatly reversed this effect.Catalpol (40 μM) downregulated the expression of NOTCH1 pathway-related proteins, such as NOTCH1, NICD1, RBPJ, HES1, and HES5, and promoted the differentiation of OPCs.Therefore, our results clearly indicated that the effect of catalpol in promoting remyelination may be related to downregulation of the expression of NOTCH1 signaling pathway-related indicators. In conclusion, catalpol exerts obvious neuroprotective effects in mice with CPZ-induced demyelination, promoting remyelination.Its mechanism may involve regulation of the NOTCH1 signaling pathway.However, further in-depth exploration of the mechanism is required. ",
"section_name": "Catalpol Promoted Remyelination by Regulating the NOTCH1 Signaling Pathway",
"section_num": null
}
] |
[
{
"section_content": "The study was supported by the National Natural Science Foundation of China (81873252, 81573898) and Beijing Natural Science Foundation (No. 7182020). ",
"section_name": "FUNDING",
"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": "The animal study was reviewed and approved by the Animal Experiments and Experimental Animal Welfare Committee of Capital Medical University (AEEI-2015-185). YS and LW designed this study.YS and JJ performed the experiments.YS, ZZ, and LW analyzed the data.YS wrote the manuscript.HZ, BX, LJ and YS revised the manuscript.All authors approved the final manuscript.LW supported the funding. 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": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "The animal study was reviewed and approved by the Animal Experiments and Experimental Animal Welfare Committee of Capital Medical University (AEEI-2015-185). ",
"section_name": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "YS and LW designed this study.YS and JJ performed the experiments.YS, ZZ, and LW analyzed the data.YS wrote the manuscript.HZ, BX, LJ and YS revised the manuscript.All authors approved the final manuscript.LW supported the funding. ",
"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
}
] |
10.1371/journal.pone.0143275
|
The chromatin regulator chd8 is a context-dependent mediator of cell survival in murine hematopoietic malignancies
|
Aberrant chromatin regulation is a frequent driver of leukemogenesis. Mutations in chromatin regulators often result in more stem-like cells that seed a bulk leukemic population. Inhibitors targeting these proteins represent an emerging class of therapeutics, and identifying further chromatin regulators that promote disease progression may result in additional drug targets. We identified the chromatin-modifying protein CHD8 as necessary for cell survival in a mouse model of BCR-Abl+ B-cell acute lymphoblastic leukemia. This disease has a poor prognosis despite treatment with kinase inhibitors targeting BCR-Abl. Although implicated as a risk factor in autism spectrum disorder and a tumor suppressor in prostate and lung cancer, the mechanism of CHD8's activity is still unclear and has never been studied in the context of hematopoietic malignancies. Here we demonstrate that depletion of CHD8 in B-ALL cells leads to cell death. While multiple B cell malignancies were dependent on CHD8 expression for survival, T cell malignancies displayed milder phenotypes upon CHD8 knockdown. In addition, ectopic expression of the Notch1 intracellular domain in a T cell malignancy partially alleviated the detrimental effect of CHD8 depletion. Our results demonstrate that CHD8 has a context-dependent role in cell survival, and its inhibition may be an effective treatment for B lymphoid malignancies.
|
[
{
"section_content": "Philadelphia-chromosome positive acute lymphoblastic leukemia (Ph+ B-ALL) accounts for approximately 20% of adult cases of leukemia in the United States [1].This disease has a poor prognosis despite the development of multiple inhibitors targeting the BCR-Abl fusion tyrosine kinase that drives this disease.Patients initially respond well to tyrosine kinase inhibitors (TKIs) but quickly relapse, usually acquiring resistance due to mutations in the Abl kinase domain that prevent TKI binding, upregulation of drug efflux pumps, or activation of alternative signaling pathways such as SRC family kinases [2][3][4].Our group previously performed a large-scale RNAi screen in a mouse model of this disease to identify factors that promote cell survival in Ph+ B-ALL and could serve as novel drug targets [5].This model expresses a human BCR-Abl transgene and a disrupted p19 ARF locus, closely recapitulating the genetics of the human disease as approximately 50% of Ph+ B-ALL patients exhibit loss-of-function of the CDKN2A/B locus that contains p19 ARF [6,7].In addition, most cells can give rise to disease in transplant experiments [6], so this model is able to represent a highly complex RNAi library in vivo. Among the list of screening hits were shRNAs targeting chromatin modifiers with established roles in cancer such as Sin3a, underscoring the importance of epigenetic regulation in leukemia progression.Inhibitors of chromatin modifiers represent an emerging class of therapeutics that holds great potential, and recent work has suggested that inhibiting these enzymes could help circumvent acquired resistance to existing drugs [8][9][10].Another chromatin modifier that arose as a candidate hit in this screen was Chd8.Chd8 has been associated with autism spectrum disorder as well as cancer, but its mechanism of action is not well understood [11][12][13][14][15][16][17].Several roles in transcriptional regulation and target gene sets have been proposed, but a consensus on the precise function of CHD8 has yet to emerge. CHD8 was discovered in a yeast two-hybrid screen as a β-catenin binding partner that inhibits transcription of β-catenin target genes [18].A proposed role of CHD8 is negative regulation of p53 and Wnt pathway activity through chromatin compaction at target genes during early embryonic development [19,20].Other studies have demonstrated a role of CHD8 in cell cycle regulation including promoting transcription of E2F target genes involved in the G1/S transition [21,22]. While multiple groups have measured higher CHD8 expression in cancer cells than normal adult tissue [19,23], other groups have observed loss of expression in gastric and colorectal cancers and deletion in lung cancer [15,16,24].It is intriguing that CHD8 appears to act in a proproliferative or pro-survival manner in most contexts but as a tumor suppressor in other malignancies, perhaps through inhibition of Wnt signaling.Further investigation is needed to determine the context in which CHD8 inhibition would be detrimental to the tumor and thus advantageous to patients.For this reason we pursued further investigation into the pro-survival function of CHD8 in BCR-Abl+ B-ALL cells. We characterized Chd8 as a pro-survival gene in this model of BCR-Abl+ B-ALL, confirming the RNAi screening results.Depletion of CHD8 resulted in cell death, but without a preceding cell cycle arrest.Interestingly, we found differing requirements for CHD8 expression between B and T cell malignancies.T-ALL cells expressing the intracellular domain of Notch (ICN) were less dependent on CHD8 expression, and ectopic expression of ICN in K-ras driven T-cell lymphoma cells partially rescued the dependency of these cells on CHD8 expression.We conclude that CHD8 is a context-dependent pro-survival factor, and that constitutive Notch signaling is able to compensate for CHD8 loss through mechanisms that are not yet fully understood. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "BCR-Abl+ B-ALL and Top Notch T-ALL cells were cultured in RPMI-1640 (HyClone) with 10% fetal bovine serum (FBS), 5μM β-mercaptoethanol, and 4mM L-glutamine.Eu-myc Arf -/- cells were cultured in 45% DMEM/45% IMDM (HyClone) with 10% FBS, 5μM β-mercaptoethanol, and 2mM L-glutamine.K-ras LA2/+ ; p53 LSL/LSL T cell lymphoma cells were cultured in IMDM with 10% FBS and 10μM β-mercaptoethanol.Pre-B cells were harvested from bone marrow of a C57BL/6 mouse, stained with fluorescently-conjugated anti-B220 (BioLegend), anti-CD11b (eBioscience), and anti-IgM antibodies (eBioscience) and sorted to obtain B220 +- CD11b -IgM -cells.Pre-B cells were cultured in 45% DMEM/45% IMDM with 10% FBS, 5μM β-mercaptoethanol, 2mM L-glutamine, recombinant murine IL-7 (1.0 ng/mL), and recombinant murine SCF (1.0 ng/mL) (Peprotech) on a feeder layer of bone marrow stromal cells. ",
"section_name": "Cell Culture",
"section_num": null
},
{
"section_content": "shRNAs were designed and cloned as previously described [25].Oligo sequences (S1 Table ) \nwere PCR-amplified with primers containing XhoI and EcoRI restriction sites (S2 Table ).shRNAs were cloned into MSCV/LTRmir30-PGK-puromycin r -IRES-GFP (MLP) or MSCV/ LTRmir30-SV40-GFP (MLS) for GFP competition assays, and TRMPVIR (TRE-dsRed-miR30/shRNA-PGK-Venus-IRES-rtTA3) for inducible shRNA studies [26].pMIG (MSCV-IRES-GFP, Addgene 9044) and pMIG-ICN were used for rescue assays.Duplin was cloned using the Gibson Assembly 1 method (New England BioLabs).mRNA was extracted from B-ALL cells using a Qiagen RNEasy kit and reverse-transcribed with the ThermoScript RT-PCR system (Life Technologies).cDNA was PCR-amplified with Phusion polymerase (New England BioLabs) with primers flanking the Duplin sequence and containing Gibson Assembly 1 overhang sequences (S2 Table ).The PCR product was ligated into modified pMIG (Not1 and Mfe1 sites inserted between EcoRI and XhoI restriction sites using oligos listed in S2 Table ).To generate retroviruses, 293T cells were transfected with plasmids using the calcium phosphate method [27]. ",
"section_name": "shRNAs and plasmids",
"section_num": null
},
{
"section_content": "Single-guide RNA sequences (S3 Table ) were designed and cloned into pSpCas9(BB)-2A-GFP according to the protocol in Ran et al [28].tdTomato+ B-ALL cells were transfected using Lipofectamine 3000 (Life Technologies) according to the manufacturer's protocol.Cells were sorted 24 hours later by GFP expression, either into 96-well plates as single cells, or into tubes and seeded into 96-well plates as single cells 3 days later.Clonal populations were analyzed for CHD8 expression by western, and editing of the Chd8 gene by sequencing (PCR and sequencing primers listed in S2 Table ).For growth competition assays, clonal populations were mixed with unlabeled control B-ALL cells and seeded in triplicate.Percentages of tdTomato+ cells were analyzed 2, 4, and 8 days later by flow cytometry.Clones Chd8-1 and -2 were generated with sgChd8-2, clone Chd8-4 was generated with sgChd8-3, and clones Chd8-3 and Chd8-5 were generated with sgChd8-4.Clones Ren-1 and -2 were generated with sgRen-2, and clone Ren-3 was generated with sgRen-3. ",
"section_name": "CRISPR-Cas9",
"section_num": null
},
{
"section_content": "Cell pellets were generated following puromycin selection (MLP) or doxycycline treatment of sorted cells (TRMPVIR).K-ras LA2/+ ; p53 LSL/LSL cells were sorted following transduction with MLS.Lysates were generated using RIPA buffer.Antibodies and dilutions are listed in S4 Table .mRNA for qPCR analysis was extracted using a Qiagen RNEasy kit and reverse-transcribed with MMLV-RT (New England BioLabs).qPCR primer sequences are listed in S5 ",
"section_name": "Western blotting and qPCR",
"section_num": null
},
{
"section_content": "Cells were partially infected with the indicated retroviruses and seeded in 6-well plates in triplicate.Percentages of GFP+ cells were determined by flow cytometry on days 2, 6, and 10 after infection.For in vivo competition assays, 2×10 6 partially infected cells were injected into female C57BL/6 (6-8 week old) mice via the tail vein.Upon disease presentation, leukemic cells were harvested from the spleen, bone marrow, and peripheral blood and analyzed by flow cytometry to determine percentages of GFP+ cells.Propidium iodide incorporation was used to exclude dead cells.For survival experiments, cells transduced with the indicated constructs were sorted by GFP expression and approximately 20 GFP+ cells per mouse (female C57BL/6, 6-8 weeks old) were injected via the tail vein.Upon disease presentation, cells were harvested from the spleen and peripheral blood and analyzed by flow cytometry to determine percentages of GFP+ cells.All mice were sourced from Jackson Laboratories. ",
"section_name": "Growth competition assays and survival experiments",
"section_num": null
},
{
"section_content": "Cells were infected with MLP, MLP-shChd8-0, or MLP-shChd8-1 and selected with puromycin.Indicated numbers of live cells were seeded in triplicate, and total numbers of live and dead cells were counted at the indicated time points by hemocytometer and trypan blue incorporation.For cell cycle analysis, B-ALL cells were infected with TRMPVIR-shRen or TRMPVIR-shChd8-1 and sorted by Venus expression.Sorted cells were plated and treated with doxycycline (Sigma, 200 ng/mL), and samples were collected at indicated time points and fixed in ethanol.Cells were stained with propidium iodide and analyzed by flow cytometry.Cell cycle profiles were created with ModFit LT software (Verity Software). ",
"section_name": "Growth curves and cell cycle analysis",
"section_num": null
},
{
"section_content": "Student's t tests, ANOVA, and survival analyses were performed with GraphPad Prism software. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the NIH.The protocol was approved by the MIT Committee on Animal Care (Protocol #0515-044-18).All efforts were made to minimize suffering.In long-term survival experiments, animals were monitored 3 times per week for two weeks (a time-frame established by prior experiments with this disease model), then daily as animals in the control cohort developed disease.Animals were euthanized when symptoms (hunched posture, lower levels of activity) were displayed.There were no unexpected animal deaths. ",
"section_name": "Ethics Statement",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To validate that the deleterious effect of the original shRNA targeting Chd8 in the screen is due to knockdown of the intended target, additional constructs targeting Chd8 were designed and tested by GFP competition assays (Fig 1A).In these assays, a population of cells is partially transduced with a retroviral vector expressing an shRNA linked to a GFP marker to allow identification of shRNA-expressing cells by flow cytometry.As with the original shRNA identified by the screen, an additional shRNA also led to a significant decrease in CHD8 expression at the protein level (Fig 1B).Consistent with an on-target effect, both constructs led to depletion of transduced B-ALL cells over time, both in in vitro and in vivo settings (Fig 1C and1D).Depletion of shChd8-expressing leukemic cells was observed in all lymphatic tissues examined (spleen, bone marrow, and peripheral blood).These results indicate that the detrimental effects of CHD8 knockdown are cell-autonomous and independent of the tumor microenvironment.Interestingly, while shChd8-0 appears to confer its deleterious phenotype more rapidly than shChd8-1 in vitro, cells expressing shChd8-1 deplete to a similar extent when examined over a longer period of time (S1 Fig). The model of BCR-Abl+ B-ALL utilized in this study is very aggressive, giving rise to terminal disease in less than two weeks following tail vein injection of 2 million cells into immunocompetent syngeneic mice.We predicted that CHD8 depletion in transplanted B-ALL cells would increase time to disease due to attenuation of cell viability and tumor growth.Cells expressing shChd8-1 or a GFP vector control were sorted to obtain pure GFP+ populations.Very low numbers of cells were transplanted by tail vein injection into each of 30 C57BL/6 mice (15 per cohort), and time to morbidity was monitored.Time to terminal disease was indeed extended significantly in mice injected with CHD8-depleted cells (Fig 1E).Disease penetrance was decreased as well, with only 2 mice in the shChd8-1 cohort succumbing to disease, compared with 10 mice in the control cohort.When mice reached morbidity, cells from the spleen and peripheral blood were collected and analyzed by flow cytometry for GFP expression.Interestingly, cells collected from mice in the control cohort were polyclonal and expressed GFP, but cells harvested from mice injected with CHD8-depleted cells were GFP-negative (Fig 1F).These results indicate that disease onset in these mice was caused by the outgrowth of a small number of GFP-negative cells contaminating the injected population. As additional confirmation that the detrimental effects of these shRNAs are due to depletion of CHD8, we performed gene editing using the CRISPR-Cas9 system.B-ALL cells expressing the tdTomato fluorophore were transfected with one of three single-guide sequences cloned into a CRISPR-Cas9 vector.Single cell clones were generated and tested for gene editing and loss of CHD8 protein expression (Figs 2A andS2).These cells were mixed with unlabeled control B-ALL cells to carry out in vitro growth competition assays.We observed depletion of tdTomato+, Chd8-edited B-ALL cells relative to control cells in these assays, confirming that loss of CHD8 is detrimental (Fig 2B).In contrast, B-ALL cells transduced with guide sequences targeting Renilla luciferase, which is not expressed in these cells, expressed normal levels of CHD8 (S3 Fig) did not deplete in similar growth competition assays (Fig 2B).In addition, mice were injected with low numbers of B-ALL cells grown from these CHD8-deficient or control clonal populations (10 mice per cohort).All 20 mice injected with control B-ALL cells succumbed to disease by Day 39 after injection, but only 6 out of 40 mice injected with cells deficient for CHD8 developed disease within 11 weeks of injection (Fig 2C). ",
"section_name": "CHD8 depletion is detrimental to growth of BCR-Abl+ B-ALL cells in vitro and in vivo",
"section_num": null
},
{
"section_content": "In order to study the downstream molecular effects of Chd8 knockdown in a temporally controlled manner, an shRNA targeting Chd8 was cloned into the doxycycline-inducible retroviral vector TRMPVIR [26].This vector utilizes a Tet-On system to control transcription of an shRNA.A reverse Tet-transactivator (rtTA) and the Venus fluorophore are constitutively expressed, and addition of doxycycline induces expression of the shRNA as well as a dsRed marker, creating a population of Venus + dsRed + cells (Fig 3A).When transduced into B-ALL cells, TRMPVIR-shChd8-1 leads to a marked reduction of CHD8 expression by 12 hours following treatment with doxycycline, while CHD8 levels in cells transduced with TRMPVIR expressing a Renilla luciferase shRNA (shRen) are not affected (Fig 3B ). assays were calculated from day 2 to day 10 after retroviral infection and normalized to an empty vector control.Fold changes of in vivo assays were calculated from day 2 after retroviral infection (day of injection) to morbidity (approximately 10 days later).In vivo assay shRNA results were compared with empty vector control in each respective tissue. ",
"section_name": "An inducible RNAi vector allows examination of gene function in a temporally controlled manner",
"section_num": null
},
{
"section_content": "Others have found that CHD8 depletion leads to a decrease in expression of CCNE2 and TYMS and a subsequent cell cycle arrest at the G1/S transition [21].We examined CCNE2 as well as CCNE1 expression by qPCR in B-ALL cells expressing TRMPVIR-shChd8-1 or the shRen control.Surprisingly, mRNA expression levels of CCNE1 and CCNE2 were found to be slightly higher in CHD8-depleted cells after 48 hours of doxycycline (Fig 3C).This result seemed counter to the expected outcome in that a decrease in cyclin expression rather than an increase would be expected to be detrimental to cell cycle progression.To determine if this change in CCNE1 and CCNE2 levels correlated with a change in cell cycle profile, cell cycle analysis was carried out on these populations.Flow cytometry analysis revealed no significant change in the cell cycle profile upon CHD8 depletion (Fig 3D ), indicating that dysregulation of CCNE1 and CCNE2 transcription upon CHD8 depletion does not lead to a G1/S arrest in B-ALL cells. However, these results do not preclude the possibility that CHD8 depletion leads to a prolonged doubling time in these cells, lengthening each stage of the cell cycle rather than arresting in a particular stage.CHD8 depletion could also lead to cell death without a preceding cell cycle arrest.To test this possibility, we retrovirally infected cells with a puromycin-selectable vector expressing one of two shChd8 constructs or a vector control.Following puromycin selection, 2.5×10 5 live cells per construct were plated in triplicate, and the total numbers of live and dead cells were quantified at 24-hour intervals.Both shChd8 constructs caused a decrease in the apparent growth rates of B-ALL cells and an increase in the percentages of dead cells (Fig 3E and3F).Taken together, these results argue that the depletion of shChd8-expressing cells observed in the growth competition assays is due to cell death rather than slowed proliferation. Notably, this death may not be due to canonical apoptosis as treatment with the pan-caspase inhibitor ZVAD-fmk simultaneously with doxycycline did not prevent CHD8 knockdownmediated cell death an in in vitro growth competition assay (S5 Fig) .In addition, no evidence of caspase 3 cleavage was seen by western blot at 12, 24, 48, or 72 hours after doxycycline treatment (S6 Fig). ",
"section_name": "CHD8 depletion leads to cell death without a preceding cell cycle arrest",
"section_num": null
},
{
"section_content": "Given that a number of CHD8 domains have been shown to interact with specific binding partners including p53, β-catenin, CTCF, and methylated H3K4 [19,20,29], we reasoned that determining the domains necessary for CHD8 function in B-ALL cells might inform important prosurvival roles.Previously, others have found that overexpression of a 110 kDa truncated N-terminal isoform of murine CHD8 called Duplin was able to rescue cells from p53-mediated apoptosis as effectively as full-length CHD8 [19].Duplin contains the first of the two chromodomains in CHD8, but not other identified functional domains [20,29].We hypothesized that overexpression of this truncated isoform might be able to rescue the shChd8 phenotype in B-ALL cells.Duplin cDNA was PCR-amplified from B-ALL cDNA and cloned into an MSCV-IRES-GFP (pMIG) retroviral vector.Transduction of B-ALL cells with pMIG-Duplin led to high expression of the protein as expected, however endogenous expression of Duplin was not observed in untransduced cells (Fig 4A).Previous studies did not measure Duplin expression in blood, so it is possible that only the full-length isoform is expressed in murine lymphoid cells.However, we were also unable to detect expression of multiple isoforms of Chd8 by qPCR in other murine tissues including brain, liver, lung, and kidney (Fig 4B).Although this isoform may not normally be expressed, we reasoned that if the domains found in Duplin are important for CHD8 function in B-ALL cells, Duplin overexpression would still be able to prevent depletion of shChd8-expressing cells in growth competition assays.shChd8-1 was selected for use in rescue experiments as it is specific for the full-length isoform.As seen in Fig 4C, exogenous expression of Duplin was unable to prevent depletion of shChd8-expressing cells, suggesting that the domains found in Duplin are not sufficient to rescue the effects of CHD8 depletion.While a full-length cDNA control that does rescue the CHD8 knockdown phenotype would make these results more conclusive, the large size of this cDNA (7.75 kb) made it challenging to clone and express in B-ALL cells despite several attempts. ",
"section_name": "Ectopic expression of CHD8 N-terminal domains",
"section_num": null
},
{
"section_content": "Previously, it had been observed that CHD8 is overexpressed in murine cancer cell lines compared to corresponding normal tissues [20].To determine if this pattern could be observed in the B-cell lineage, bone marrow was harvested from healthy adult C57BL/6 mice, and pre-B cells (B220 + IgM -CD11b -) were collected by flow cytometry.Unexpectedly, CHD8 expression in these untransformed pre-B cells was comparable to that seen in B-ALL cells (Fig 5A).The dependence of these cells on CHD8 expression was also tested by a growth competition assay in vitro.While protein expression levels in these cells may be similar, biological differences between the cell types might make untransformed pre-B cells less reliant on CHD8 for survival.However, untransformed pre-B cells expressing shChd8 depleted in vitro to a similar extent as B-ALL cells, indicating that in the B lymphoid lineage dependence on CHD8 expression is not limited to malignant cells (Fig 5B). ",
"section_name": "Untransformed pre-B cells require CHD8 for survival",
"section_num": null
},
{
"section_content": "While CHD8 expression is required in untransformed pre-B cells, malignancies with different genetic backgrounds or within different developmental contexts may have mechanisms that allow cell survival in the absence of this protein.We reasoned that identifying a context-specific survival requirement could shed light on CHD8 function.To determine whether dependency on CHD8 extends to other hematopoietic malignancies, we first tested another cancer of the B cell lineage, a murine model of Burkitt's lymphoma.In this model, c-myc is under the transcriptional control of the μ immunoglobulin heavy chain enhancer (Eμ-myc), mimicking the t (8;14)(q24;q32) chromosomal translocation that causes this disease in humans [30][31][32].We transduced these cells with shRNAs targeting Chd8 and confirmed decreased protein expression by western blot (Fig 5C).As with BCR-Abl+ B-ALL cells, Eμ-myc cells expressing these shRNAs deplete in in vitro growth competition assays (Fig 5D ), and pure populations of shRNA-expressing cells display decreased proliferation rates and increased numbers of dead cells compared with cells expressing vector controls (Fig 5E and5F). A recent study into vulnerabilities of drug-resistant T-ALL cells uncovered a number of epigenetic regulators that became essential upon acquisition of drug resistance [8].CHD8 was one of several epigenetic regulators found to be preferentially required for survival by γ-secretase inhibitor (GSI)-resistant cells over syngeneic, GSI-sensitive cells.These resistant cells exhibited a lower level of Notch signaling than the sensitive population, suggesting that high Notch pathway activity may somehow compensate for CHD8 loss.In agreement with these results, we found that a T-ALL cell line that constitutively expresses the intracellular domain of Notch (\"Top Notch\") [33], and thus exhibits constitutive activation of the Notch pathway, is less dependent on CHD8 expression for survival than the B cell malignancies tested.Top Notch cells expressing shChd8-1 did not deplete in in vitro growth competition assays (Fig 6A).While shChd8-0 caused depletion that was determined to be statistically significant, this depletion was less than that seen in B-ALL cells. We also examined CHD8 dependency in a spontaneous T-cell lymphoma cell line derived from a K-ras LA2/+ ; p53 LSL/LSL mouse (hereafter referred to as KP).These cells express oncogenic K-ras G12D and are functionally p53-null [34,35].KP lymphoma cells deplete upon CHD8 knockdown in growth competition assays, but to a lesser extent than B cell malignancies (Fig 6B).The distinction between CHD8 dependencies in these two T cell populations could shed light on the mechanism of CHD8 activity.To determine if constitutive Notch signaling could rescue the CHD8 knockdown phenotype, KP lymphoma cells were transduced with the Notch intracellular domain (ICN) linked to a GFP marker, or a GFP marker alone (S7 Fig) [36].Ectopic ICN expression is lethal to B cells [37], so they could not be used for these experiments.Transduced KP cells were sorted to obtain pure GFP+ populations, then partially transduced with one of two shChd8 constructs linked to a tdTomato marker, or a vector control.Cells were analyzed by flow cytometry to assess the change in percentage of tdTomato+ cells in each population.While cells expressing shChd8 on the GFP+ control background depleted to a significant extent, cells expressing shChd8 on the ICN-transduced background were partially rescued from depletion (Fig 6C).This result indicates that constitutive Notch signaling can attenuate the detrimental effects of CHD8 knockdown in KP lymphoma cells, possibly overriding a reduction in survival or growth signals caused by CHD8 depletion that other oncogenic signaling pathways are unable to compensate for. ",
"section_name": "Differential requirement for CHD8 expression in hematopoietic malignancies",
"section_num": null
},
{
"section_content": "We have demonstrated that CHD8 is necessary for survival of B lymphoid malignancies.This dependency exists in both the in vitro and in vivo settings, indicating that CHD8 functions in a cell-autonomous manner and its depletion does not alter interactions of B-ALL cells with their microenvironment.When we examined the effects of CHD8 depletion on leukemic cell proliferation, we uncovered a cell death phenotype not preceded by cell cycle arrest.Others have shown that CHD8 knockdown in cervical carcinoma cells leads to G1 arrest facilitated by downregulation of CCNE2 and TYMS, genes required for transition into S phase [21].However, when we examined cell cycle profiles of B-ALL cells upon CHD8 knockdown, we observed no difference between control and CHD8-depleted cells.It is possible that the differences between our results and those of Rodriguez-Paredes et al. are due to the distinct signaling contexts in the two cell types used. Several early studies observed expression of a truncated N-terminal CHD8 isoform termed Duplin [18,19].We found that exogenous expression of Duplin did not compensate for CHD8 knockdown, suggesting that domains unique to full-length CHD8 are crucial for its function in B-ALL cells.The second chromodomain, absent in Duplin, has been shown to enable binding to histone H3 [21], and the BRK domains at the C-terminus have been demonstrated to interact with the chromatin insulator CTCF [29].It is curious that, unlike results of previous studies, endogenous Duplin expression was not found in B-ALL cells or in normal mouse tissues by qPCR.Bioinformatics databases currently show truncated isoforms of CHD8 only in rat, so it is likely that murine Duplin is an experimental artifact not physiologically expressed. Previous work by our group has highlighted genes whose effect on survival is context-specific between B and T cell malignancies [5].For example, the chromatin regulator PHF6 is required for survival of B-ALL cells, but loss-of-function is selected for in T-ALL [38].Factors such as HES1 that promote T lineage development are often lethal when ectopically expressed in the B cell lineage [37].We found that Top Notch T-ALL cells are significantly less dependent on CHD8 expression than the B cell malignancies tested.The results of the ICN rescue experiment indicate that constitutive Notch signaling can partially compensate for CHD8 knockdown in a T cell malignancy, consistent with results of a recent screen for chromatin regulators that are essential in GSI-resistant cells [8].CHD8 was among the proteins found to be necessary for survival of resistant cells but not syngeneic GSI-sensitive cells that continued to exhibit high levels of ICN and a more open chromatin conformation.These results suggest that CHD8 and high Notch signaling are able to compensate for each other's absence through mechanisms that are currently unclear.It is possible that CHD8 compensates for chromatin compaction upon Notch inhibition by promoting transcription.Alternatively, CHD8-mediated chromatin compaction could promote survival of GSI-resistant cells that have downregulated Notch signaling.Additional investigation is needed to determine the lineage-specific mechanism of CHD8's pro-survival activity. A growing body of literature shows that inhibition of chromatin-modifying proteins is a promising field of investigation and drug development.Inhibiting these factors may correct global transcriptional deregulation instigated by events such as Myc overexpression.Our work suggests CHD8 is a potential drug target in B cell malignancies provided toxicity in normal hematopoietic cells is not limiting.Our results and those of others indicate that inhibiting CHD8 would not be as effective in T cell malignancies driven by Notch signaling, and could be counter-productive in certain solid tumors.However, CHD8 inhibition could synergize with γsecretase inhibitors in cells that have become resistant through epigenetic mechanisms.When compared to our knowledge of other chromatin-modifying proteins, our understanding of the function of CHD8 is relatively incomplete.Nonetheless, it is clear that CHD8 has critical roles in cell survival, and additional investigation should be conducted to better define its place within central signaling pathways. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "The authors thank Charles Sherr for BCR-Abl+ B-ALL cells, Anthony Capobianco for Top Notch T-ALL cells, Tyler Jacks for K-ras LA2/+ ; p53 LSL/LSL T-cell lymphoma cells, and Warren Pear for pMIG-ICN. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "This work was funded by the Ludwig Foundation for Cancer research (http://www.ludwigcancerresearch.org).JRS was funded by by an NSF graduate Fellowship.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
}
] |
10.1186/s12874-022-01728-0
|
Improving outcomes for patients with lymphoma: design and development of the Australian and New Zealand Lymphoma and Related Diseases Registry
|
<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>Lymphoma is a malignancy of lymphocytes and lymphoid tissues comprising a heterogeneous group of diseases, with up to 80 entities now described. Lymphoma is the 6<jats:sup>th</jats:sup> most common cancer in Australia, affecting patients of all ages, with rising incidence rates. With the proliferation of efficacious novel agents, therapeutic strategies are increasingly diverse and survival is improving. There is a clear need for contemporary robust and detailed data on diagnostic, investigational and management strategies for this disease in Australia, New Zealand and worldwide, to inform and benchmark local and international standards of care. Clinical quality registries can provide these data, and support development of strategies to address variations in management, including serving as platforms for clinical trials and other research activities. The Lymphoma and Related Diseases Registry (LaRDR) was developed to capture details of patient demographics, disease characteristics, and management throughout their disease course and therapy and to develop outcome benchmarks nationally and internationally for lymphoma. This report describes the aims, development and implementation of the LaRDR, as well as challenges addressed in the process.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>The LaRDR was established in 2016 as a multicentre, collaborative project at sites across Australia with a secure online database which collects prospective data on patients with a new diagnosis of lymphoma or chronic lymphocytic leukaemia (CLL). LaRDR development required multidisciplinary participation including specialist haematology, information technology, and biostatistical support, as well as secure funding. Here we describe the database development, data entry, ethics approval process, registry governance and support for participating sites and the coordinating centre.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>To date more than 5,300 patients have been enrolled from 28 sites in Australia and New Zealand. Multiple challenges arose during the development, which we describe, along with approaches used to overcome them. Several confirmed international collaborations are now in place, and the registry is providing valuable data for clinicians, researchers, industry and government, including through presentations of results at major national and international conferences.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusion</jats:title> <jats:p>Challenges in establishing the LaRDR have been successfully overcome and the registry is now a valuable resource for lymphoma clinicians, researchers, health economists and others in Australia, New Zealand and globally.</jats:p> </jats:sec>
|
[
{
"section_content": "The Lymphoma and Related Diseases Registry (LaRDR) was established in 2016 with the aim to improve the quality of care and clinical outcomes for people with lymphoma and chronic lymphocytic leukaemia (CLL), through systematic collection, analysis and reporting of real-world data to understand lymphoma epidemiology, current management and outcomes in Australia and New Zealand.Here we describe the rationale, development process and initial experience from the registry. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "Clinical quality registries (CQR) are now well established internationally, and endorsed as integral to continuous improvement in healthcare through supporting delivery of and monitoring evidence-based practice [1].Key features of CQRs and their value, including for blood cancers, are reviewed in detail elsewhere [2][3][4].By collecting a standard minimum dataset, which increasingly includes patient-reported outcomes, registries are also very valuable for uncommon diseases or interventions where clinical trials are challenging, and even large referral centres may see few patients.In this context registries can provide a mechanism to identify variation in practice.They also and serve as efficient platforms to conduct observational studies and interventional trials to establish optimal management and conduct health economics analyses using 'real world' data [2][3][4].Linkage with other datasets, such as cancer and death registries, can also be readily undertaken. ",
"section_name": "The value of clinical registries",
"section_num": null
},
{
"section_content": "Lymphomas are cancers of lymphocytes and lymphoid tissues -the lymph nodes and related organs, such as the spleen.These cancers are classified according to their cell of origin and increasingly by molecular diagnostics, with more than 80 entities now recognised [5].Lymphoma is the sixth most common cancer diagnosis in Australia with more than 6000 new diagnoses annually, and the incidence is rising [6].CLL is the single most common lymphoid cancer in adults with over 2000 new cases reported annually in Australia.Its long natural history of asymptomatic disease, with many never requiring treatment, and unique features compared with other lymphoid cancers highlighted the desirability of a CLLspecific module (see below). Lymphoid malignancies affect people of all ages, and impose a significant burden for patients and the health system, with high rates of hospitalisations for treatment delivery and for management of complications, such as infection [6].Therapies are often complex, and must be tailored to the specific type of lymphoid cancer with many patients undergoing multiple lines of therapy during the course of their disease; management may include a combination of chemotherapy, immunotherapy, small molecule drugs, radiation, cellular therapies such as autologous or allogeneic haematopoietic stem cell transplant or chimeric antigen-receptor T-cell therapeutics, and occasionally surgery, along with supportive care measures such as immunoglobulin replacement therapy and transfusions.Survival is improving likely due to improvements in diagnosis, better supportive care, and the availability of new targeted therapies, but many of these are costly, and also carry specific adverse effect profiles. Few Australian data are available on lymphoma treatments and outcomes outside the setting of clinical trials, and fewer than 5% of adult cancer patients are enrolled on clinical trials [7].State cancer diagnosis registries can provide important but limited data on diagnoses and deaths, but no information on patient factors such as comorbidities, treatment or outcomes other than death, including quality of life.CQRs can help address many of the substantial evidence gaps that need to be addressed to better inform policy and improve practice and outcomes. With increasing complexity of diagnosis and management, a need was identified for contemporary national Australian epidemiological, therapy, clinical outcome and health economic data for lymphoma and CLL to complement clinical trials, and a lymphoma CQR was proposed.LaRDR is managed by the School of Public Health and Preventive Medicine at Monash University, a large academic organisation with expertise in clinical quality registries, in partnership with participating hospitals and clinicians.Site investigators oversee activities at participating hospitals.A multidisciplinary project team (project managers, data managers, registry experts, lymphoma clinician) coordinates day-to-day activities, and provides support to the steering committee and site staff and investigators. ",
"section_name": "A clinical quality registry for lymphoma",
"section_num": null
},
{
"section_content": "The registry is supported by multiple industry partners, on a sponsorship and/or project basis.These partners can request targeted analyses and reports based on their interests, but do not direct the overall research activities of the registry.Industry funding is acknowledged as a potential conflict of interest in presentations and publications.A modest per patient payment to sites supports data entry activities. ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "The LaRDR has human research ethics committee (HREC) approval from Monash Health (HREC 16/ MonH/74) and all participating hospital sites, now (since 2016) under a national mutual acceptance (NMA) ethics scheme which allows publicly funded health services across all jurisdictions to accept an ethical review from an external accredited HREC.NMA arrangements were in place for clinical trials but not for registries at the time of commencing work on LaRDR, necessitating time-consuming HREC applications to all initial sites individually.Local governance approvals are still required to ensure sites can support the project activity.In 2022, the registry expanded to New Zealand following approval by the New Zealand Health and Disability Ethics Committees (reference: 2022 FULL 12203, 29 March 2022). LaRDR utilises an \"opt out\" consent model, an approach approved by the Australian National Health and Medical Research Council and New Zealand National Ethics Advisory Committee if the public interest in a research study sufficiently outweighs the potential impingement on individual privacy.This model enables maximum participation and thereby reduces bias; it is widely used for registry activities in Australia.Clinicians at participating sites are responsible for identifying potential participants, explaining the study to them, inviting them to participate, and providing them the approved LaRDR information brochure, which describes the registry aims, data being collected and LaRDR contact details.This process is documented in the patient's file in the registry.No written consent is required.Patients may opt out at any time from initial invitation or in the future, at which point any of that person's data will be deleted centrally.The consent also provides for centralised review of laboratory results and histology slides. Registry analyses by approved investigators using existing LaRDR data can be conducted without additional HREC approval.Sub-studies requesting additional data typically require additional approval. The project is registered on the Australian and New Zealand Clinical Trials Registry (ACTRN12617000050358). ",
"section_name": "Ethics approval and consent to participate",
"section_num": null
},
{
"section_content": "Patients 18 years or older, with diagnoses of any type of non-Hodgkin lymphoma, Hodgkin lymphoma, CLL or related diseases in accordance with the WHO classification [5], are eligible to participate.The registry collects prospective data on incident cases -a case being defined as having received a diagnosis subsequent to or within 6 months prior to the participating site securing HREC approval to participate in LaRDR, in order to minimise selection bias and the burden of retrospective data collection and to maximise data completeness.An exception is made for CLL, which is frequently slowly progressive, and a significant proportion of patients may never require CLL-directed therapy.Therefore, retrospective data on CLL patients diagnosed up to 10 years previously can be included, provided complete data are available.For deceased patients where the cause of death is listed as lymphoma or CLL a waiver of consent is in place to obtain data. ",
"section_name": "Patient selection",
"section_num": null
},
{
"section_content": "Data items included in the minimum dataset are listed in Table 1.Datasets and case report forms for lymphoma and CLL were designed by the steering committee and project team, and refined iteratively.A CLL-specific case report form was developed due to the particular staging, disease trajectory and therapeutic paradigm applicable to this disease subtype.Data dictionaries are available for reference. The minimum data set includes information on demographics, comorbidities, diagnosis, planned therapy (if any) and supportive care, which are collected at baseline, with relevant updates plus disease response and survival entered at 6 and 12 months, and annually thereafter.Quality of life and biobanking data options were included to accommodate future projects.Data items are added (or deleted if not needed or feasible to collect) with approval of the Steering Committee and LaRDR data manager. ",
"section_name": "Establishing a minimum dataset",
"section_num": null
},
{
"section_content": "LaRDR uses a REDCap database hosted and managed by Helix at Monash University.REDCap (Research Electronic Data Capture) is a secure, web-based software platform designed to support data capture for research studies, providing 1) an intuitive interface for validated data capture; 2) audit trails for tracking data manipulation and export procedures; 3) automated export procedures for seamless data downloads to common statistical packages; and 4) procedures for data integration and interoperability with external sources [8,9]. The database has a user-friendly interface and requires only basic training for site staff.To minimise data entry error, and aid analysis, most fields were designed to be dropdown, check boxes or radio buttons with minimum free text requirements, since data collection is typically performed by non-medically trained staff who rely on hospital electronic and paper patient medical records and may not be familiar with specific disease-or treatmentrelated details.Clarification on specific items can be sought from lymphoma and CLL experts on the project team, and/or site investigators. LaRDR project staff conduct quality control activities, review data queries, and provide feedback and reports to site staff, investigators and the steering committee.A data validation committee reviews inconsistencies to refine definitions, data fields and user instructions, and conducts audits to review data completeness and accuracy.Sites may access their own data at any time and can manage local reports to facilitate local audits and data completion. Students, medical specialists in training and others undertake research using registry data.All research projects must be approved by the steering committee who provide oversight to all approved projects, ensuring no overlap between projects and to help ensure timely completion.In accordance with the LaRDR data access policy, projects that require patient-level data access this via Monash University's secure environment for sharing research data (SeRP), a secure platform that allows researchers to analyse de-identified, patient-level data.Results from these analyses must first be approved by the data custodian before they can be exported, with only aggregate data approved for export.LaRDR staff are available to provide statistical support as well clinical insight to all projects.Data are published in an aggregate form. Data linkage with state and national cancer registry data are planned to ensure that all eligible patients at participating sites are captured, and that missing or discrepant cases are followed up with sites.Annual linkage with the National Death Index in each country is planned to validate survival data. ",
"section_name": "Data management, quality control, and analysis",
"section_num": null
},
{
"section_content": "1. Pathology review working group: There are over 80 recognised subtypes of lymphoma with distinct biol- and management has now diverged significantly from non-Hodgkin lymphoma.Not all patients require treatment, but for those who do, the optimal use of newer therapies, including combinations and sequencing of agents, is yet to be defined.Furthermore emerging evidence supporting a key prognostic role for genetic and measurable residual disease testing in this condition requires an evidence base to support its optimal clinical application.A dedicated working group comprising 11 CLL experts designed the CLL-specific dataset and data fields, which was integrated into the existing LaRDR database and tested before being made accessible to other registry users.3. Data validation committee: The management of lymphoma is rapidly evolving, with new treatment protocols and diagnostic tests continually emerging.The role of the data validation committee, made up of lymphoma and registry experts, is to ensure the registry keeps pace with this evolution by reviewing the data fields that are collected and updating them as appropriate. ",
"section_name": "Working groups",
"section_num": null
},
{
"section_content": "Hospital data reports are provided annually to individual sites, with site-specific, aggregate de-identified patient data presented and compared with overall national data.A breakdown of major diagnostic groups and their characteristics, treatment and survival data, and information on data completeness, is included.This allows benchmarking with other health services nationally and participating hospitals can identify site-specific issues for clinical audit and further investigation.Sites with low patient recruitment receive generic reports until sufficient data have accrued (see example: Additional file 1). Summary LaRDR annual reports are published on the LaRDR website (lardr.org).Annual open meetings, usually conducted in conjunction with the national haematology scientific congresses, or virtually in 2020-21, provide opportunities for clinicians, site staff, industry partners and students to learn more about the registry.Scientific results are presented at local and international conferences and published in the peer-reviewed literature [10][11][12][13].Commissioned reports are also provided to industry partners and may be requested by others (for example, government agencies). ",
"section_name": "Communications and reporting",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "The registry commenced with a pilot in 2016 with 6 large metropolitan hospitals in Australia with lymphoma expertise and resources and who had expressed interest in participating.These sites and their teams were crucial in planning, testing and providing feedback on all aspects of the registry, including governance and operations, and refining the minimum dataset, data entry processes and the database.Data completeness reports were generated and fields with low completion rates reported back to data managers and compared with detailed information from site staff on data that were onerous to find in medical records or where instructions were unclear.Results were discussed by the steering committee and a number of important changes made to the database and processes based on this feedback.An indicative timeline of registry establishment and progress is given in Fig. 1. ",
"section_name": "Pilot phase and activities",
"section_num": null
},
{
"section_content": "Across six Australian states and two territories, and one New Zealand site, more than 5300 patients are currently enrolled from 28 sites, with 33 hospitals open to recruitment and a further 2 sites awaiting governance approval; others have also expressed interest in joining.Whilst still dominated by large tertiary metropolitan centres, site profiles are diversifying, with the addition of five regional hospitals and one private hospital since the pilot phase.Recruitment to date is shown in Fig. 2 and the frequency of cases according to major disease group in Table 2. National Australian coverage is currently estimated at 20% of lymphoma cases diagnosed annually, and continuing to expand.All sites in Australia and New Zealand are now welcome to join the registry. Some key diagnostic and demographic data are presented in Table 2. Median follow-up time for prospectively enrolled patients on the registry is 18 months, with 35% of patients having more than two-years follow-up.As the data mature, follow-up times will increase which is important given the long disease course and excellent prognosis of some lymphoma subtypes and CLL.The first data linkage with the Victorian Cancer registry is currently underway, and we plan to begin annual linkages with the National Death Index to validate mortality data and improve estimates of overall survival, a key endpoint in many analyses. The registry has already generated interest among the international lymphoma clinical and patient communities, government, and industry partners.To date, 20 research projects have been completed or are underway using registry data and infrastructure, with three international collaborations now formalised, delivery of 26 national and international conference presentations, and provision of 20 data reports to industry, investigators and government, along with publication of a manuscript (see: lardr.org/ resea rch/# Resea rchpu blica tions and [10][11][12][13]). ",
"section_name": "Current status",
"section_num": null
},
{
"section_content": "LaRDR is now established and delivering new national data on lymphoma and CLL epidemiology, management and outcomes.By describing and sharing our experiences, we hope that this will assist others planning similar activities, as we ourselves have built on the experience of the project team and investigators, including previously setting up the Australian and New Zealand Myeloma and Related Diseases Registry and other registries [4].Some of the challenges with establishing LaRDR are applicable to all registries (and many other types of major research infrastructure projects).These are summarised in Table 3.Other aspects of the early LaRDR experience also hold lessons for establishing registries for other complex conditions.These include confirming the initial diagnosis: lymphoma diagnosis and staging is complex, requiring a set of coordinated clinical, imaging and pathology investigations, some of which are specific to particular disease subtypes.Even expert pathologists can disagree on diagnostic assignments in lymphoma, and this is even more challenging in a national registry setting since it is not practical to require review of primary diagnostic material for all cases from every site -and there would be implications for changing a diagnosis after subsequent review where the patient has already received therapy.Managing the diversity of diagnoses (and accounting for changing diagnoses where low-grade diseases transform to a more aggressive form), and periodic updates to the WHO classification, with implications for existing and new entries in the database, adds further complexity.Furthermore, the registry was established to collect data prospectively, intending to enhance data completeness and reduce bias.However, CLL and some types of lymphoma are typically very indolent in their disease course, with little change in status over years or even decades.Patients with stable CLL managed with 'watch and wait' approach are not captured in clinical trials (as by definition they do not require treatment), but nevertheless have disease complications such as immune Highlighting advantages for: • Patients: Knowing their data will help create a national picture of the condition, and that their hospital is participating in benchmarking for best practice failure, and autoimmune disease.These patients can also be markedly under-represented in registry data.The long-term follow-up is also important to capture complications such as second malignancy that tend to occur more commonly over time [14].Furthermore, with the dramatic improvements over the last decade with immunochemotherapy and then novel therapies, prolonged survival is much more commonly seen than previously and these long-term complications related to this improved survival will be important to document as the 'new natural history' of CLL.To address these important questions, the CLL group allowed retrospective data entry for CLL where sites were confident of access to complete data. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "LaRDR is now an established CQR.It is well placed to continue its expansion with increased national coverage in both Australia and New Zealand, and to support future research, including by publishing results of analyses and providing epidemiological data (such as numbers and geographic location of patients with data on diagnoses and disease stage) which will inform planning of clinical trials.The registry can also serve as a platform for conducting clinical trials [15] and observational studies, and enable efficient, long-term follow up after these studies have been completed.In addition to information contained within the registry itself, LaRDR data can be used for epidemiological modelling and linkage activities to inform policy development and health service planning, especially for new and high-cost therapies and to ensure improved access to and delivery of care for all patients. ",
"section_name": "Future directions",
"section_num": null
}
] |
[
{
"section_content": "The authors thank all patients participating in the Lymphoma and Related Diseases Registry.We acknowledge the site staff for their efforts. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "The Lymphoma and Related Diseases Registry has received funding from Abbvie, Antengene, AstraZeneca, Beigene, Bristol Myers Squibb, Celgene, Gilead, Janssen, Novartis, Roche, Sandoz and Takeda. ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "Access to data that support the findings of this study are available from the Lymphoma and Related Diseases Registry with permission from the Steering Committee and in accordance with the LaRDR Data Access Policy.More information is available at lardr.org or via email to: SPHPM-Lymph oma@ monash.edu ",
"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/ s12874-022-01728-0.• 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 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/ s12874-022-01728-0.• thorough peer review by experienced researchers in your field ",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "",
"section_name": "Additional file 1.",
"section_num": null
},
{
"section_content": "• 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": "Steering committee",
"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/s12943-022-01698-3
|
Extracellular vesicle microRNAs contribute to Notch signaling pathway in T-Cell Acute Lymphoblastic Leukemia
|
<jats:title>Abstract</jats:title><jats:p>T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive T-cell malignancy characterized by genotypically-defined and phenotypically divergent cell populations, governed by adaptive landscapes. Clonal expansions are associated to genetic and epigenetic events, and modulation of external stimuli that affect the hierarchical structure of subclones and support the dynamics of leukemic subsets. Recently, small extracellular vesicles (sEV) such exosomes were also shown to play a role in leukemia. Here, by coupling miRNome, bulk and single cell transcriptome profiling, we found that T-ALL-secreted sEV contain NOTCH1-dependent microRNAs (EV-miRs), which control oncogenic pathways acting as autocrine stimuli and ultimately promoting the expansion/survival of highly proliferative cell subsets of human T-cell leukemias. Of interest, we found that NOTCH1-dependent EV-miRs mostly comprised members of miR-17-92a cluster and paralogues, which rescued in vitro the proliferation of T-ALL cells blocked by γ-secretase inhibitors (GSI) and regulate network of genes characterizing patients with relapsed/refractory early T-cell progenitor (ETP) ALLs. All these findings suggest that NOTCH1 dependent EV-miRs may sustain the growth/survival of immunophenotypically defined cell populations, altering the cell heterogeneity and the dynamics of T-cell leukemias in response to conventional therapies.</jats:p>
|
[
{
"section_content": "Acute lymphoblastic leukemia or ALL, is an aggressive malignancy of immature lymphocytes with about 15-20% of cases of T lineage (T-ALL).It is the most common type of cancer in children, but also affects adults with incidences of ~ 30 new cases per 1,000,000 per year [1]. Pediatric T-ALL is largely curable with intensive chemotherapy, but there are significant side effects and ~ 20% of patients suffer relapse.In contrast, adult T-ALL is characterized by a 5-year overall survival of ~ 40% [2]. T-ALL is the result of a malignant alteration of hematopoietic progenitors during T-cell development.A relevant oncogenic pathway involved in T-cell transformation is the NOTCH1 signaling pathway with over 50% of human T-ALL carrying activating mutations of NOTCH1 gene [3,4]. Recently, small extracellular vesicles (sEV) such as exosomes were reported to contribute to leukemic progression [5,6].sEV were shown to reprogram the bonemarrow microenvironment [7], dampen anti-leukemia immune response [8] and promote drug resistance [9].sEV exert such molecular and cellular functions by transferring molecular information from cancer cells to proximal and/or to distant body districts, including prometastatic niche [10].Importantly, in T-ALL a miRNAtumor suppressor gene network drives the malignant transformation of T-cell progenitors [11,12] and cooperates with NOTCH1-driven T-ALL [13][14][15][16][17].However, the precise role of sEV and miRNA cargo in NOTCH1driven T-ALL remains elusive.Here, we tackle this issue and present new evidences supporting a central role for EV-miRs in the progression of NOTCH1-driven T-ALL. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "The molecular characteristics of sEV in T-ALL were initially explored in CUTLL1 cell line, a well-characterized human T-cell lymphoma cell line derived from a pleural effusion in a pediatric patient with T-ALL at relapse, with aberrant NOTCH1 activation and strongly sensitive to γ-secretase inhibitors [18].CUTLL1 cells were lentivirally transduced to constitutively express a dominantnegative form of Mastermind-like protein 1 (dnMAM) to shutdown NOTCH1 signalling, or an empty vector as a control.Indeed, several NOTCH1 target genes were strongly reduced under dnMAM condition (Fig. 1A,B; Fig. S1A).Next, we analyzed size distribution, morphology, quantities of sEV released from CUTLL1-CTRL and CUTLL1-dnMAM cells by nanoparticle-tracking (NTA), TEM, and WB analyses using sEV markers (Fig. 1C-D; Fig. S1B-C).Overall, the prevalent size of sEV matched with expected exosome size distribution (i.e., ~ 30-150 nm; Fig. 1C) and sEV concentration was significantly increased in dnMAM cells (Fig. 1C) in line with previous results showing that a deranged NOTCH signaling in T cells induces a dramatic increase in exosomes release [19].We detected a total of 318 miR-NAs (Fig. 1E; Table S1) by whole-miRNA expression profiling of CUTTL1 cells of which 73 also detected in sEV (i.e., Common-miRs; Fig. 1E; Table S1).Yet, hierarchical clustering analysis showed a set of highly abundant 'EV-miRs' comprising members of miR-17-92a cluster and paralogues (Fig. 1F), which we found to be characterized by overrepresented EXOmotifs (Figure S1D), i.e. sorting sequences that determine miRNAs upload into sEV [20,21].In line with previous reports, miR-19b is highly expressed in T-ALL cells and is targeted by the t(13;14) (q32;q11) translocation in T-ALL.Likewise, other members of miR-17-92a clusters i.e., miR-20a and miR-92a, were found highly expressed in T-ALL and together with miR-19b were shown being capable of promoting T-ALL [11].Expression profiling analysis of the Common-miRs in T-ALL cells and in sEV (Fig. 1E), revealed a significant and specific decreased expression of miR-17-92 cluster and paralogues upon NOTCH1 signalling inactivation (i.e., dnMAM vs. CTRL; Fig. 1G) both intracellularly and in sEV (Fig. 1G), which suggests NOTCH1 signalling modulates EV-miRs quantities in sEV.As matter of fact, previous studies showed the existence of a tight interplay between c-MYC and miR-17-92 cluster expression [22] and that c-MYC is an important direct target of Notch-1 in T-ALL [23].Consistently, we found that miR-17-92 cluster expression is rescued in dnMAM condition upon forced expression of c-MYC (Fig. S1E-G).Next, to investigate the function of these NOTCH1-dependent (See figure on next page.)Fig. 1 EV-miRNAs characterization and function in T-ALL model.A ddPCR analysis of validated NOTCH1 target genes mRNA expression in CUTLL1-dnMAM vs. CUTLL1-CTRL cells.Y-axes, mRNA levels of NOTCH1 target genes normalized to B2M expression.X-axes, gene symbols.Significance analysis was performed by one-sample t-test.B Immunoblot analysis of HES1, HES4 and c-MYC proteins in CUTLL1-CTRL and CUTLL1-dnMAM cells.C Nanoparticle-tracking-analysis of the size distribution and concentration of sEV released by CUTLL1-CTRL and CUTLL1-dnMAM cells.Inset plots, Transmission Electron Microscope (TEM) images showing particles in sEV samples from CUTLL1-CTRL and CUTLL1-dnMAM cells.Scale bar = 100 nm.On the right, box plots of differential concentration of sEV in CUTLL1-CTRL and CUTLL1-dnMAM cells.Significance analysis was performed by Student t-test.D Immunoblot analysis of sEV markers (CD81, Syntetin1 and CD63) in CUTLL1-CTRL and CUTLL1-dnMAM cells.E Venn diagram of EV-miRNAs detected in CUTLL1-CTRL and CUTLL1-dnMAM cells (CELL) or in their released small extracellular-vesicles (sEV).Significance analysis was performed by Fisher's exact test.F Hierarchical clustering analysis of miRNAs detected (N = 318) in CUTLL1-CTRL (Ctrl) and CUTLL1-dnMAM (dnM) cells (CELL) and/or in their released small extracellular-vesicles (sEV).On the right, most abundant miRNAs in sEV were also indicated; in bold, members of the miR-17-92 cluster.G On top, violin plots of differential expression (dnMAM vs. CTRL) of the 73 commonly detected miRNAs in CUTTL1 cells and in sEV.Bottom, bar plots of differential expression (dnMAM vs. CTRL) of the miR-17-92 cluster and paralogues.Colors are as per the legend.Significance analysis was performed by Mann-Whitney U-test.H qRT-PCR analysis of miR-17-92 cluster overexpressing CUTLL1-dnMAM cells vs. control (Empty-V) CUTLL1-dnMAM cells.Significance analysis was performed by one-sample t-test.I ddPCR analysis of miR-17-92 cluster in sEV purified from miR-17-92 cluster overexpressing vs. control (Empty-V) CUTLL1-dnMAM cells.Bubble size represents the average expression of miRNAs (copies/20µL).Colours are as per the legend.J Flow cytometry analysis of CUTTL1 (CTRL) and CUTTL1-dnMAM cells (dnMAM) conditioned with PKH26-labelled miR-17-92-enriched sEV (EV_miR-17-92) or PKH26-labelled Empty-Vector sEV (EV_Empty-V) derived from miR-17-92 overexpressing CUTTL1 cells or from CUTLL1 cells transfected with an empty vector, respectively.MFI, mean fluorescence intensity.Percentages of cells which internalized exogenous PKH26-sEV (Cells EV-pos) are also shown.K Viability of CUTTL1 (CTRL) and CUTTL1-dnMAM cells (dnMAM).Briefly, transduced GFP positive cells were FACS sorted and in vitro grown together with miR-17-92-enriched sEV (EV_miR-17-92) or sEV (EV_Empty-V) cultured for two days.GFP + alive cells were measured by flow cytometry for DAPI (4′,6-diamidino-2-phenylindole) exclusion and counted by relating the cell numbers to internal fluorescent bead events (see also methods).The graph reports the result of two independent experiments.Significance analysis was performed by Student's t-test EV-miRs, we produced PKH26-labelled sEV enriched in miR-17-92 (aka EV_miR-17-92) by overexpressing miR-17-92 cluster in dnMAM cells which yielded sEV enriched in miR-17-92 cluster (Fig. 1H-I; see methods).Internalization of EV_miR-17-92 in dnMAM cells (Fig. 1J) significantly increased the proliferation rate to a comparable level to NOTCH1-proficient CUTLL1 CTRL cells (Fig. 1K).We then treated CUTLL1-wt cells with γ-secretase inhibitor (GSI; see supplemental methods) and observed, as expected, a strong impairment of cell viability (Fig. S2A).Contrariwise, EV_miR-17-92 induced expansion of CUTLL1-wt cells (p < 0.01; Student's T-test; Fig. 2A) and, importantly, were able to rescue the GSI-induced phenotype in T-ALL cells (Fig. 2A).Similar results were obtained by using cells from two independent clones of T-ALL patient-derived xenografts (PDX) (Fig. S2B).Of note, using a known NOTCH1dependent miRNA i.e. the miR-223-3p (Table S1; [24,25]) we obtained comparable results in vitro (Fig. S2C-E).Finally, leukemia cells of M71 and H3255 PDX lines were transduced with lentiviruses encoding miR-17-92 cluster or empty vector (EV) as control and subsequently transplanted into immunocompromised (NSG) mice.In line with all previous results, we observed that miR-17-92 transduced human cells indeed rescued the GSI-induced phenotype in T-ALL PDXs (Fig. 2A-B). Taken together, such results showed, for the first time, the ability of sEV_miR-17-92 to propagate molecular information among T-ALL cells which was able to restore, at least in part, a defective NOTCH1 signalling pathway. Lastly, we dissected the molecular function of miR-17-92 cluster in the realm of NOTCH1-driven T-ALL.We reasoned that NOTCH1 signalling can be generalized in two main routes: Path-A) the 'canonical' transcriptional output of NOTCH1 intracellular domain (NCID) (Fig. 2C); Path-B) the transcriptional output controlled by NOTCH1 through miR-17-92 (Fig. 2C).High-throughput gene expression profiling of CUTLL1 cells ± miR-17-92, and GSI/mock treated (Fig. 2D; see methods) followed by quantitative trait analysis (see methods) identified two transcriptional gene modules, i.e.Mod-A (N = 966 genes) and Mod-B (N = 765 genes), which differ in terms of transcriptional regulation and are both dependent to GSI treatment yet indifferent to rescued miR-17-92 expression (i.e., Mod-A; Fig. 2D; see methods), or reverted (i.e., Mod-B Fig. 2D) (Table S2).Such results confirmed our hypothesis of a bipartite NOTCH1 signalling transcriptional output (Fig. 2C).Notably, MSigDB analysis of Mod-B gene sets revealed a strong and significant enrichment (FDR q-value < 0.0001) of predicted miR-17-92-targeted transcripts (Table S3; see methods) further confirming the regulatory function of the miR-17-92 cluster in Mod-B.Furthermore, IPA software (see methods) revealed that Mod-A comprised canonical NOTCH-signalling genes (e.g., NOTCH2-4, c-MYC, CTNNB1, GATA1-3, etc.) (Fig. S3A) while Mod-B was enriched in gene involved in proliferation (CDKN2A, CCNE1, E2F3, E2F6, RBL1), stemness (FOXM1, TCF4) and cancer (ETS1, RELA, NFE2L2) (Fig. S3B).Intriguingly, when we used Mod-A and Mod-B gene sets to stratify an external cohort of human T-ALL (i.e., the Liu et al. cohort, N = 261; Table S4; [26]), we observed that Mod-B gene set hallmarks T-ALLs particularly enriched in the Early T-cell precursor (ETP) and pre-/post-cortical subtypes, with a higher post-therapeutic minimal residual disease (MRD), and blast count in the bone marrow, that are all characteristics of an adverse outcome [27][28][29](Fig.2E-F; Table S5). Next, we performed single-cell RNA sequencing of primary cells, derived from T-ALL patients (N = 2), without any expansion in vivo into immunocompromised mice.Using the Phenograph algorithm [30], we identified several distinct cell subsets (n = 11) (Fig. 2G).Gene set enrichment analysis (GSEA) using Mod-A and -B and hierarchical clustering analysis revealed three main clusters grouping T-ALL cell subsets which shared similar pattern of enrichment scores (ES) (Fig. 2H).In particular, CL2 contains cell subsets contributed by both two patients (Fig. S2F-G) with coherent expression trend of both Mod-A and -B as defined in Fig. 2D, which is a hallmark of activity of NOTCH1 signalling pathway.Indeed, GSEA using Hallmark gene sets (see methods) confirmed that these CL2-cell subsets were significantly characterized by mechanisms involved in proliferation and metabolism (Fig. 2I), which further show how both canonical' NCID signalling (Mod-A) and NOTCH1 miR-17-92 mediated signalling (Mod-B) can contribute to Notch1related phenotypes and coexist in the same T-ALL cell subsets. Our findings shed new light on composite interactions between sEV-miRs, Notch signalling and cellular plasticity that characterize the tumor heterogeneity of T-ALL and promote relapsed/refractory cell subsets of T-cell leukemias.In this scenario, further investigations are needed to explore such mechanisms in T-ALL with the final intent of offering more efficient therapies targeting diverse oncogenic states and microenvironments that support aggressive tumor cells. ",
"section_name": "Results and discussion",
"section_num": null
},
{
"section_content": "For extensive details on all methodologies see online Supplemental Material and Methods. ",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "The institutional ethical committees approved this study (registration number: N91/CE), and informed consent was obtained from all patients enrolled. ",
"section_name": "Human samples",
"section_num": null
},
{
"section_content": "Expression levels of 754 miRNAs were quantified using the TaqMan Human MicroRNA Array A + B Card Set v3.0 (Applied Biosystems, Foster City, CA). ",
"section_name": "Profiling by TaqMan Human MicroRNA Arrays",
"section_num": null
},
{
"section_content": "Gene expression profiling was performed using the Gene-Chip ® Human Clarion S Array (Thermo Fisher Scientific) including more than 210,000 distinct probes representative of > 20,000 well-annotated genes (hg19; Genome Reference Consortium Human Build 37 (GRCh37)). ",
"section_name": "Genome-wide expression profiling",
"section_num": null
},
{
"section_content": "Whole transcriptome analysis at single cell level was performed on FACS-sorted primary T-ALL cells using the BD Rhapsody Single-Cell Analysis System (BD, Biosciences). ",
"section_name": "Single cell RNA-sequencing (scRNA-Seq)",
"section_num": null
}
] |
[
{
"section_content": "We are grateful to Chiara Di Giorgio for critically editing the manuscript and Dr. Rossella Di Paola for her help in the Illumina next generation DNA sequencing.We would like to thank Dr. Andrew P. Weng (BC Cancer research, Terry Fox Laboratory) for providing us with PDX samples of T-ALL.We also thank Dr. Alessandra Aloisi and Dr. Riccardo Dicorato (CNR-IMM, Lecce Unit) for support with TEM analysis.This study was performed in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of \"Casa Sollievo della Sofferenza\" Foundation.All authors gave their consent to publication.The gene expression data used in this study are publicly available as indicated in the Methods and Supplementary Information sections. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This work was in part supported by the Associazione Italiana Ricerca sul Cancro [IG-22827 to F.B.; IG-23070 to V.G.], the Italian Ministry of Health [GR-2016-02363975 and CLEARLY to F.B.; GR-2016-02361287 to V.G; GR-2019-12370460 to T.C.; Ricerca Corrente program 2022-2024 to F.B. and to V.G.], Worldwide Cancer Research [20-0318 to V.G].T.C. was supported by a fellowship from the Associazione Italiana Ricerca sul Cancro (#19548) and the Umberto Veronesi Foundation.F.T. was supported by a fellowship for Italy from the Associazione ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "The normalized (U6) data for miRNA can be found in Table S1 while mRNA expression data can be accessible at NCBI GEO (GSE193482) and SRA (PRJNA784728 for scRNA-Seq data). The normalized (U6) data for miRNA can be found in Table S1 while mRNA expression data can be accessible at NCBI GEO (GSE193482; reviewer token: gjubkewovfsvdyr) and SRA (PRJNA784728 for scRNA-Seq data). ",
"section_name": "Data set availability",
"section_num": null
},
{
"section_content": "The normalized (U6) data for miRNA can be found in Table S1 while mRNA expression data can be accessible at NCBI GEO (GSE193482) and SRA (PRJNA784728 for scRNA-Seq data). ",
"section_name": "Data set availability",
"section_num": null
},
{
"section_content": "The normalized (U6) data for miRNA can be found in Table S1 while mRNA expression data can be accessible at NCBI GEO (GSE193482; reviewer token: gjubkewovfsvdyr) and SRA (PRJNA784728 for scRNA-Seq data). ",
"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/ s12943-022-01698-3. Additional file 2. Additional file 4. Supplemental materials and methods.S1. miRNA normalized (U6) expression data.Table S2.Mod-A and Mod-B gene sets.Table S3.MSigDB analysis of Mod-B genes.Table S4.Liu et al.T-ALL cohort stratified according to ssGSEA analysis performed with Mod-A and Mod-B gene sets (N=261 patients; see supplemental materials and methods).Table S5.Clinicopathologic characteristics of Liu et al.T-ALL cohort stratified according to ssGSEA analysis performed with Mod-A and Mod-B gene sets (N=261 patients; see supplemental materials and methods). Conception and design: TC, VG, FB; Development of methodology: TC, PP, EDS, FT, VM, RC, GR, VG, FB; Acquisition of data: TC, PP, FT; Analysis and interpretation of data: TC, PP, VG, FB; Writing, review, and/or revision of the manuscript: TC, VG, FB; Administrative, technical, or material support: PP, GR; Study supervision: VG, FB.The author(s) read and approved the final manuscript. The institutional ethical committees approved this study (registration number: N91/CE), and informed consent was obtained from all patients enrolled. All authors have agreed to publish this manuscript. The authors declare that there is no conflict of interest. • 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 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": "The online version contains supplementary material available at https:// doi.org/ 10. 1186/ s12943-022-01698-3. ",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "Additional file 2. ",
"section_name": "Additional file 1.",
"section_num": null
},
{
"section_content": "Additional file 4. Supplemental materials and methods.S1. miRNA normalized (U6) expression data.Table S2.Mod-A and Mod-B gene sets.Table S3.MSigDB analysis of Mod-B genes.Table S4.Liu et al.T-ALL cohort stratified according to ssGSEA analysis performed with Mod-A and Mod-B gene sets (N=261 patients; see supplemental materials and methods).Table S5.Clinicopathologic characteristics of Liu et al.T-ALL cohort stratified according to ssGSEA analysis performed with Mod-A and Mod-B gene sets (N=261 patients; see supplemental materials and methods). ",
"section_name": "Additional file 3.",
"section_num": null
},
{
"section_content": "",
"section_name": "Additional file 5: Table",
"section_num": null
},
{
"section_content": "Conception and design: TC, VG, FB; Development of methodology: TC, PP, EDS, FT, VM, RC, GR, VG, FB; Acquisition of data: TC, PP, FT; Analysis and interpretation of data: TC, PP, VG, FB; Writing, review, and/or revision of the manuscript: TC, VG, FB; Administrative, technical, or material support: PP, GR; Study supervision: VG, FB.The author(s) read and approved the final manuscript. ",
"section_name": "Authors' contributions",
"section_num": null
},
{
"section_content": "The institutional ethical committees approved this study (registration number: N91/CE), and informed consent was obtained from all patients enrolled. ",
"section_name": "Declarations Ethics approval and consent to participate",
"section_num": null
},
{
"section_content": "All authors have agreed to publish this manuscript. ",
"section_name": "Consent for publication",
"section_num": null
},
{
"section_content": "The authors declare that there is no conflict of interest. • 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": "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/s41467-021-23752-2
|
Targeted PI3K/AKT-hyperactivation induces cell death in chronic lymphocytic leukemia
|
<jats:title>Abstract</jats:title><jats:p>Current therapeutic approaches for chronic lymphocytic leukemia (CLL) focus on the suppression of oncogenic kinase signaling. Here, we test the hypothesis that targeted hyperactivation of the phosphatidylinositol-3-phosphate/AKT (PI3K/AKT)-signaling pathway may be leveraged to trigger CLL cell death. Though counterintuitive, our data show that genetic hyperactivation of PI3K/AKT-signaling or blocking the activity of the inhibitory phosphatase SH2-containing-inositol-5′-phosphatase-1 (SHIP1) induces acute cell death in CLL cells. Our mechanistic studies reveal that increased AKT activity upon inhibition of SHIP1 leads to increased mitochondrial respiration and causes excessive accumulation of reactive oxygen species (ROS), resulting in cell death in CLL with immunogenic features. Our results demonstrate that CLL cells critically depend on mechanisms to fine-tune PI3K/AKT activity, allowing sustained proliferation and survival but avoid ROS-induced cell death and suggest transient SHIP1-inhibition as an unexpectedly promising concept for CLL therapy.</jats:p>
|
[
{
"section_content": "ignaling derived from the B-cell surface immunoglobulin, the B-cell receptor (BCR), generally promotes the survival and proliferation of B cells.However, throughout their development, B cells are selected depending on their binding capacity to the respective antigen at several B-cell selection checkpoints.While B cells that express a functional BCR, which induces adequate BCR signaling, are positively selected by promoting B-cell survival and expansion, B cells lacking a sufficient BCR signal die of neglect 1,2 .These checkpoints also safeguard against autoimmunity by clonal deletion of autoreactive B cells that exhibit excessive BCR-signaling strength.Upon constitutively strong autoantigen binding, B cells undergo an active negative selection process that eliminates or inactivates autoreactive B-cell clones 3 .B cells are therefore selected for a narrow window of intermediate strength of BCR signaling, since both too weak (no functional BCR) and excessive strength (autoreactive BCR) of BCR signaling results in the clonal deletion and cell death.Several oncogenic drivers in B-cell malignancies constitutively activate BCR signaling and thereby mimic signaling that promotes positive selection and B-cell expansion 4 . In chronic lymphocytic leukemia (CLL), autonomous autoreactive BCR signaling contributes to tumor cell survival 5 , and established targeted therapy concepts focus on inhibiting oncogenic kinases in the BCR pathway [6][7][8] , resulting in signal deprivation and thereby cell death 9,10 .However, despite initial remission of CLL, patients frequently relapse with refractory disease or eventually progress to Richter syndrome transformation with limited treatment options 11 .Recent evidence in pre-Bcell acute lymphoblastic leukemia (pre-B ALL) suggests that targeted hyperactivation of signaling components downstream of the BCR above a maximum threshold will also invariably trigger cell death [12][13][14] .This indicates that, despite the transformation, malignant B cells remain vulnerable to checkpoint signals for removal of autoreactive clones.While direct agonists of BCRdownstream kinases are not available, targeted hyperactivation can be achieved by pharmacological inhibition of negative regulators of the BCR-signaling pathway (e.g., inhibitory phosphatases including SHIP1), resulting in the activation of signaling above a maximum tolerable threshold causing energy stress and cell death.This effect likely occurs because pre-B ALL cells are in an early developmental B-cell stage, where B cells are subject to central tolerance checkpoints for removal of autoreactive clones 15 .As exemplified in pre-B ALL, pharmacological hyperactivation of BCR signaling may represent a powerful strategy to overcome conventional drug resistance and to prevent relapse induced by long-term kinase inhibitor treatment. While in many B-cell malignancies, the activation of the BCRsignaling pathway occurs via genetic activation of signaling mediators 16,17 or by BCR mimicry 18,19 , the BCRs expressed by CLL cells frequently recognize autoantigens, including an internal epitope of their own BCR 5 .The activation of the BCR-signaling pathway occurs primarily in secondary lymphoid organs 20 .Here, it typically converges with activation of the phosphatidylinositol-3-kinase (PI3K) signaling pathway downstream of homing chemokine receptors and adhesion molecules 21,22 , with the PI3Kδ isoform as the most prevalent PI3K subclass in mature B cells 23 .Upon its recruitment and activation by binding to phosphorylated YxxM motifs in membrane coreceptors, such as CD19 or BCAP in case of BCR stimulation, PI3K then phosphorylates the lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] to generate phosphatidylinositol-3,4,5-trisphosphate [PI (3,4,5)P 3 ], which acts as a pivotal second messenger signaling molecule by providing a binding site for intracellular enzymes that contain pleckstrin homology (PH) domains.For instance, AKT requires binding with its PH domain to PI(3,4,5)P 3 to become enzymatically active.The AKT1 isoform in CLL cells plays an important role in driving cell proliferation, growth, survival, and cellular metabolism 24,25 . The PI3K signaling is negatively regulated by the SH2containing inositol 5′-phosphatase SHIP1 that hydrolyzes PI (3,4,5)P 3 to PI(3,4)P 2 , thereby preventing the recruitment and activation of PH domain-containing effectors and the propagation of PI3K-mediated downstream signals 26 .Mutations causing hyperactivation of the PI3K pathway are among the most common genetic lesions in human cancer 27 , and somatic mutations in the INPP5D gene encoding SHIP1 have been detected in acute myeloid leukemia (AML) patients 28 .These mutations strongly reduce SHIP1 activity, either by directly interfering with the enzymatic activity of SHIP1 to suppress PI3K/AKT signaling, or by loss of function of their SH2 domain or PXXP motifs, both of which are required for proper recruitment of the inhibitory complex 29 .Similarly, frame-shifts, as well as other translationallyinactivating deletions and insertions in the INPP5D gene, occur in T-cell acute lymphoblastic leukemia (T-ALL) 30 .In strong contrast, SHIP1 inhibition or genetic deletion of INPP5D in BCR-ABL1-driven pre-B ALL mimics excessively strong signaling from an autoreactive BCR and engages a B-cell intrinsic negative selection program leading to energy stress and cell death 12,14 .Pre-B ALL cells are derived from B-cell precursors that are subject to central B-cell tolerance checkpoints.CLL cells exhibit a mature Bcell phenotype and are thought to be derived from naïve B cells.Here we tested whether CLL cells-like pre-B ALL cells-are subject to mechanisms of negative B-cell selection.Previous studies have suggested that SHIP1 is expressed in CLL; 31 however, the functional role of SHIP1 in limiting PI3K signaling in established CLL is still not clearly defined. In this study, we, therefore, investigated the cellular consequences of acute AKT activation and SHIP1 inhibition in CLL in vitro and in vivo.While intermediate levels of PI3K/AKT activity are essential for the survival of CLL cells, we show that the negative regulator SHIP1 is required to balance PI3K/AKT signaling in CLL to prevent hyperactivation of BCR-downstream signaling and clonal deletion.Accordingly, we propose transient SHIP1 phosphatase inhibition as a potential therapeutic option and a promising strategy to overcome mechanisms of drug resistance in CLL. ",
"section_name": "S",
"section_num": null
},
{
"section_content": "Acute AKT1 hyperactivation in CLL is detrimental to CLL cells.In order to investigate how CLL cells respond to acute activation of the PI3K/AKT signaling pathway, we transduced the CLL-derived cell line MEC-1 with a constitutively active form of AKT1 (myrAKT1 32 ) linked with GFP.Unexpectedly, we observed a decrease in myrAKT1-expressing GFP + MEC-1 cells over time as compared to the untransduced, GFP-negative cells while the fraction of GFP empty vector (EV) control cells remained stable in the culture (Fig. 1a,b).AKT overexpression and activation was confirmed using western blot in myrAKT1-expressing MEC-1 cells as compared to EV transduced cells sorted for GFP (Fig. 1c).These results indicate that acute activation of the PI3K/ AKT signaling pathway diminishes CLL cell viability and expansion in this model and thereby contradict the current understanding of a solely proliferative and prosurvival role of AKT1 activation in CLL. To extend these findings in primary CLL cells, we employed the TCL1 transgenic (TCL1tg) CLL mouse model 33 .For this, we first created a transgenic mouse line for inducible AKT1 E17K expression, a mutation derived from solid tumors and T-cell lymphoma 34 that renders AKT1 constitutively active by increasing the affinity for PI(3,4,5)P 3 and confering affinity to the abundant plasma membrane lipid PI(4,5)P 2 , which is not bound by AKT1 under normal circumstances 35 .We introduced the human AKT1 E17K cDNA preceded by a loxP-flanked transcriptional and translational STOP cassette into the ubiquitously expressed Rosa26 locus, followed by an IRES GFP cassette to track AKT1 E17K -expressing cells 36 (Supplementary Fig. 1a-c).The resulting Rosa26 loxSTOPlox AKT1 E17K mice were crossed with Mb1-CreER T2 transgenic animals for B-cell-specific inducible Cre expression 37 .The STOP cassette is excised upon tamoxifen (4-OHT) treatment specifically in the B-cell-lineage, and AKT1 E17K expression is indicated by GFP expression.After crossing the AKT1 E17K Mb1-CreER T2 line to the TCL1tg CLL model, we obtained triple transgenic animals carrying TCL1tg AKT1 E17K Mb1-CreER T2 and waited for CLL development in these mice without activating the AKT1 E17K transgene.Once signs of disease occurred, we harvested splenocytes of the TCL1tg AKT1 E17K Mb1-CreER T2 mice and confirmed CLL development by detecting a pronounced CD19 + CD5 + CLL cell population in the spleen that did not express the AKT1 transgene, as indicated by the GFP-negative CLL/B-cell population (Fig. 1d).As a control for normal, non-transformed B cells, we isolated splenocytes from a littermate carrying the AKT1 E17K transgene and Mb1-CreER T2 but not the TCL1 transgene (TCL1wt).We then tested for the AKT1 E17K /GFP expression by administering 4-OHT in vitro. While both non-transformed B cells and CLL cells recombined the locus and expressed AKT1 E17K as indicated by GFP expression, AKT1 E17K promoted B-cell survival only in nontransformed B cells in vitro, while the GFP + CLL cells rapidly decreased and died in vitro (Fig. 1e,f).Next, we investigated whether the inducible activation of AKT1 E17K affects murine CLL growth in vivo.To this end, we transplanted splenocytes of aged TCL1tg AKT1 E17K Mb1-CreER T2 mice into wild-type (wt) recipients and induced AKT1 E17K expression by providing tamoxifen-enriched chow or control chow for 4 weeks.We followed the CLL content in the peripheral blood and observed a significant drop in the CLL content of the tamoxifen-treated group post transplantation (Fig. 1g).However, no GFP + CLL cells were detected in the peripheral blood suggesting that AKT1 E17K expression is not tolerated in murine CLL cells as dying cells are rapidly cleared and therefore not detectable in vivo 38 (Fig. 1h).To exclude the possibility that AKT1 E17K -expressing CLL cells relocate to the secondary lymphoid organs, we harvested spleens and lymph nodes 8 weeks post transplantation.We found a moderate decrease in the spleen size as well as decreased lymph node sizes in the tamoxifen-treated group as compared to the control mice (Fig. 1i-k).When we performed flow cytometric analyses for CD19, CD5, and GFP expression, we observed a moderate reduction in CLL in the lymph nodes (Fig. 1l) but not in the spleen (Fig. 1m).Importantly, we did not detect a substantial percentage of GFP + CLL cells in the lymphoid organs despite tamoxifen treatment.Therefore, the vast majority of CLL in the tamoxifen-treated mice did not express GFP/AKT1 E17K , suggesting that virtually all CLL cells that recombined the locus to express AKT1 E17K had died and were cleared in vivo 38 , which is in line with our in vitro studies.However, there was a small but clearly visible GFP + population detectable in the peritoneal cavity and the lymph node of tamoxifen-treated mice (less than 0.2% of CLL cells, summarized in Supplementary Fig. 1d,e) but not in the control mice, demonstrating that the genetic rearrangement had occurred upon tamoxifen treatment but did not result in CLL cell expansion.Taken together, we, therefore, conclude that acute hyperactivation of the PI3K/AKT pathway by introducing constitutively active AKT1 in CLL is detrimental for the cells in vitro and in vivo. The inhibitory phosphatase SHIP1 is highly expressed and active in CLL.We next identified strategies to therapeutically exploit the sensitivity of CLL cells to PI3K/AKT hyperactivation.As direct activators of PI3-kinases for pharmacological studies are not available, we investigated whether targeted hyperactivation can be achieved by pharmacologically inhibiting the inhibitory phosphatase SHIP1.To determine whether SHIP1 inhibition could present a useful approach in CLL, we first investigated SHIP1 expression and activity in CLL samples.The analysis of mRNA expression levels of INPP5D, encoding the phosphatase SHIP1, revealed that all 210 CLL samples analyzed 39 expressed SHIP1 mRNA, with higher levels in samples derived from CLL patients with mutated IgV H genes that have a favorable clinical prognosis as compared to those with an unmutated IgV H (Fig. 2a).However, there is no significant association of SHIP1 mRNA expression with CLL patients' time to treatment or overall survival (Supplementary Fig. 2a).We then analyzed SHIP1 protein levels of primary CLL samples and compared them to B cells of healthy age-matched donors, both derived from the peripheral blood, and found significantly higher SHIP1 expression and phosphorylation levels in CLL than in normal peripheral B cells, which is potentially induced by the activated BCR status in CLL 5 (Fig. 2b).CLL patients' characteristics for all samples used in this study are listed in Supplementary Table 1.In AML, recurrent Fig. 1 Acute AKT1 activation is detrimental to CLL cells of murine and human origin.a Time course FACS analysis of GFP expression in MEC-1 cells upon transduction with pMIG EV (empty vector) or pMIG-myrAKT1, representative for 2 independent experiments.The gating strategies are shown in Supplementary Fig. S5.b Fold change of GFP expression in MEC-1 Eco cells upon transduction with pMIG-EV (black squares) or pMIG-myrAKT1 (red squares), pooled analysis of 2 independent experiments.c Immunoblot confirmation of active AKT expression in GFP-sorted MEC-1 Eco cells upon transduction with pMIG-EV or pMIG-myrAKT1, representative for 2 independent analyses.DHX9 serves as a loading control (LC).d Splenocytes from aged TCL1wt AKT1 E17K Mb1-CreER T2 (top) or TCL1tg AKT1 E17K Mb1-CreER T2 (bottom) mice were analyzed for CD19 and CD5 expression (CLL development).GFP negativity of murine B cells (mBc) in the upper panel and murine CLL (mCLL) in the lower panel was confirmed (to exclude potential leakiness of the CreER T2 system).Representative data for n = 3 (CLL) and n = 2 (mBc) mice.e GFP expression was confirmed in both normal B cells (upper panel, GFP blot is gated for viable CD19 + CD5 -cells) and CLL cells (lower panel, GFP blot is gated for viable CD19 + CD5 + cells) upon TAT-Cre or 4-OHT administration, indicating AKT1 E17K transgene expression (left panels).4 days later, GFP + normal murine B cells (mBc; upper panel) were slightly enriched while GFP + murine CLL cells (mCLL; lower panel) significantly declined (right panels) over time.Representative data for n = 3 (mCLL) and n = 2 (mBc) mice.f The differential response to AKT1 E17K expression on the viability of mBc (black squares) and mCLL (red squares) by induction of AKT1 E17K on cell viability is shown over time (representative data are shown in top panel) and a summary of three independent experiments from n = 3 (CLL) and n = 2 (mBc) biologically independent mice (d4-8, depending on TAT-Cre or 4-OHT induction; lower panel).Data are presented as individual values and mean values ± standard deviation (SD).Statistical significance was assessed by a two-tailed unpaired Student's t-test.g-m Splenocytes from an aged TCL1tg AKT1 E17K Mb1-CreER T2 mouse were transplanted into 10 wt recipients and provided with tamoxifen-containing chow (TAM; clear squares; n = 5 individual mice) or control chow (control; black squares; n = 5 individual mice) for 4 weeks, thereafter all mice received control chow.On d7 and d14, CLL content was monitored in the peripheral blood by flow cytometric analysis of CD19 + CD5 + cells.Data are presented as individual values and mean values ± SD, statistical significance was assessed by a two-tailed unpaired Student's t-test; (n.s.) not significant (g).Representative example of GFP analysis for CLL in the peripheral blood (PB) on d14 post injection and tamoxifen (TAM) or control chow administration (h).Macroscopic analysis of lymph nodes (axillary) and spleens 8 weeks post transplantation.Scale bars indicate 1 cm.(i).Lymph node (j) and spleen size (k) are shown, determined by area quantification using the ImageJ software.Percent CD19 + CD5 + CLL cells in the lymph nodes (l) and spleen (m) of wt mice 8 weeks post transplantation is shown (j), for n = 5 individual animals, respectively.Data are presented as individual values and mean values ± SD and statistical significance was assessed by a twotailed unpaired Student's t-test in j-m; Source data are provided as a Source Data file. mutations in the SHIP1-encoding gene INPP5D lead to a significantly reduced phosphatase activity 29 .To determine whether recurrent loss-of-function mutations in the SHIP1-encoding INPP5D gene also exist in CLL, we next identified the types and frequency of INPP5D alterations in CLL in publically available sequencing data [40][41][42] (using the cBioportal for Cancer Genomics platform (http://cbioportal.org))and compared them to INPP5D mutations previously found to reduce SHIP1 phosphatase activity 29 or protein stability 43 (6 mutations detected in 1048 analyzed CLL samples).Of the 6 mutations, none occurred recurrently or was previously described as inactivating the SHIP1 phosphatase activity (listed in Supplementary Fig. 2b).Thus, INPP5D gene alterations are rare in CLL suggesting that SHIP1 is enzymatically active in the vast majority of CLL cases.To further confirm that the enzymatic activity of SHIP1 was stable in primary CLL samples, we performed a malachite green phosphate assay after SHIP1 precipitation.In primary CLL samples, we found variable but detectable levels of PI(3,4,5)P 3 dephosphorylating activity in all samples comparable to control cells that lentivirally overexpressed wt SHIP1.In healthy donor B-cell samples, SHIP1 activity was below the detection limit in 3 out of 5 tested donors, possibly due to the low expression levels in the absence of additional stimuli (Fig. 2c and Supplementary Fig. 2c).Taken together, we, therefore, conclude that CLL cells express high levels of enzymatically active SHIP1. SHIP1 inhibition induces AKT activation and is toxic specifically for CLL cells.To investigate the functional relevance of SHIP1 phosphatase activity in CLL, we first tested a small molecule SHIP1 inhibitor 3AC (3 α-Aminocholestane) that selectively inhibits the enzymatic activity of SHIP1 (IC 50 ~2.5 μM) but not related phosphatases SHIP2 and PTEN (IC 50 > 1 mM) 44 .To confirm that SHIP1 inhibition hyperactivates the PI3K/AKT signaling pathway in CLL, we analyzed AKT S473phosphorylation levels and, as expected 45 , found significantly increased activation upon SHIP1 inhibition (Fig. 2d).Further analyses of downstream events indicated the transient activation of the mTOR/S6 signaling pathway upon 3AC treatment with upregulation of the anti-apoptotic protein MCL1, followed by a decrease below baseline levels (Supplementary Fig. 2d).At continuous treatment for 48 h, the SHIP1 inhibitor induced dosedependent cell death in all 28 primary CLL samples tested (Fig. 2e).To account for the variation in the vehicle-treated control cells due to spontaneous apoptosis of primary CLL cells in vitro, we calculated the specific cell death induced by 3AC 46 .In order to determine whether the cytotoxic effects were specific for CLL, we compared the effects of 3AC on the viability of nonmalignant B cells purified from the peripheral blood of healthy donors, as well as several B-cell lymphoma cell lines, namely BJAB, SUDHL6, and Bal17, to primary CLL samples and the CLL-like cell lines MEC-1 and EHEB.While B cells derived from the peripheral blood of healthy donors and Bal17, BJAB and SUDHL6 lymphoma cells remained largely unaffected in terms of viability upon 3AC treatment (Fig. 2f), SHIP1 inhibition-induced cell death specifically in CLL cells, including the MEC-1 and EHEB cell lines.Amongst CLL samples, cells derived from patients with a favorable prognosis (mutated IgV H ) were more sensitive to SHIP1 inhibition than those with a poor prognosis (expressing unmutated IgV H ; Supplementary Fig. 2e).In order to further clarify whether the activation of AKT is critical for 3ACmediated cytotoxicity, we treated MEC-1 cells with the AKT inhibitor AZD-5363 and evaluated cell viability upon SHIP1 inhibition.Strikingly, co-treatment with the AKT inhibitor significantly reduced the cytotoxic effect of the SHIP1 inhibitor 3AC (Fig. 2g), while AKT inhibition alone had no significant effect on cell viability (Supplementary Fig. 2f).This was also confirmed in samples derived from the peripheral blood of seven individual CLL patients (Fig. 2h).Together, this data demonstrates that SHIP1 inhibition mediates cytotoxicity specifically in CLL via AKT activation. ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To test whether transient inactivation of SHIP1 is a potential therapeutic option for CLL, we assessed disease progression in different in vivo models of CLL.To determine the effects of SHIP1 inhibition on CLL in an immunocompetent system, we again used the TCL1tg CLL mouse model 33 , which is an established tool for studying therapeutic targets in human CLL 47 .Here, we evaluated the disease progression in the peripheral blood upon transplantation of murine donor CLL cells (mCLL) that revealed a relatively indolent progression in vivo and initiated the 3AC treatment when the CLL fraction was clearly detectable in the peripheral blood (d8, Fig. 3a).The treatment schedule is shown in Supplementary Fig. 3a.We then followed the expansion of CLL cells in the peripheral blood over time and observed a significant reduction in the CLL progression in the SHIP1 inhibitor-treated mice (Fig. 3b).We then analyzed the CLL infiltration in the secondary lymphoid organs 30 days post CLL injection.All CLL target organs revealed significantly less CLL cell infiltration in the 3AC-treated group as compared to the control group (Fig. 3c).To also mimic therapeutic approaches in CLL patients that already have high levels of CLL in the peripheral blood at the time of treatment initiation, we also tested the efficacy of SHIP1 inhibition in a highly aggressive mCLL where the disease had progressed to ~50% CD19 + CD5 + cells in the peripheral blood at the time of treatment initiation (Fig. 3d).Similar to our previous results, treatment with 3AC diminished the progression of the disease in the peripheral blood (Fig. 3e) and reduced the levels of remaining CLL in all target organs of murine CLL, despite the short treatment schedule of 8 doses (Fig. 3f, treatment schedule is shown in Supplementary Fig. S3b).To also assess the efficacy of SHIP1 inhibition on human CLL in vivo, we treated NOD/ SCIDcγ -/-(NSG) mice xenografted with primary patientderived CLL cells with the SHIP1-specific inhibitor 3AC in vivo (treatment schedule shown in Supplementary Fig. 3c).The gating strategy for the analysis of primary CLL cells is depicted in Supplementary Fig. 3d.Equal engraftment of hCLL cells was confirmed prior to injection (Fig. 3g).Similar to the treatment of murine CLL, we observed a significant reduction in the amount of hCLL cells in CLL target organs in the 3AC treatment group as compared to the control group, determined by flow cytometric analysis (Fig. 3h,i) and histology (using immunohistochemistry detecting human CD20 in the spleen, Fig. 3j,k).The HE staining of the histology with hCD20 analysis and labeling for statistical evaluation of sections derived from five mice per group is shown in Supplementary Fig. 3e.The SHIP1-specific inhibitor 3AC did not induce toxicity in vivo as indicated by the constant body weight of the treated mice pre-and post-treament (Supplementary Fig. 3f).Together, these results reveal that transient pharmacological SHIP1 inhibition represents a potential therapeutic strategy for treating murine and human CLL in vivo. Genetic validation of effects mediated by SHIP1 inhibition.In order to validate on-target effects of SHIP1 inhibitors mediating the observed cytotoxic effects, we employed genetic loss-offunction studies.We first performed shRNA knockdown experiments using the CLL-derived cell line MEC-1.We confirmed reduced SHIP1 protein levels with two INPP5D-targeting vectors (shSHIP1 KD1 and KD2) as compared to scrambled shRNA control cells with immunoblot (Fig. 4a).When analyzing the viability of MEC-1 cells with SHIP1 knockdown, we observed a reduction in viability (Fig. 4b) and cell count upon SHIP1 knockdown (Fig. 4c), similar to the effects observed upon 3AC treatment.To investigate the relevance of SHIP1 expression in vivo, we transplanted MEC-1 cells carrying scrambled control shRNA or SHIP1-targeting shRNA into NSG mice, a model for aggressive, rapidly progressing human CLL 48 .Based on the different viability rates at the time of cell injection, we used luciferase-expressing MEC-1 cells that can be detected via bioimaging in vivo.We conducted bioimaging on the day of cell injection to confirm similar amounts of viable MEC-1 cells in all transplanted mice (Fig. 4d, top panel).However, over a period of 4 weeks, SHIP1 knockdown significantly reduced the MEC-1 expansion in vivo as indicated by a reduced light signal detected via bioimaging (Fig. 4d, lower panels).MEC-1 SHIP1 knockdown-bearing mice lived significantly longer than those that received MEC-1 cells with scrambled shRNA vectors (Fig. 4d,e).Importantly, reanalysis of SHIP1-targeting shRNA carrying MEC-1 cells obtained from mice with active disease revealed that these expressed SHIP1 at levels similar to those in the control MEC-1 cells, indicating that the clones that escaped the knockdown outgrew and formed lethal leukemia (Fig. 4f).In addition, we performed CRISPR/Cas9-mediated knockout of the INPP5D gene in MEC-1 cells.After successful validation of targeting through western blot (Fig. 4g) and gene sequencing (Supplementary Fig. 3g), we performed competitor growth assays and confirmed the selective disadvantage of SHIP1-deficient MEC-1 cells in vitro (Fig. 4h) and a similar trend was observed in vivo (Fig. 4i).Note that SHIP1 knockout MEC-1 cells were expanded from single cells for several weeks prior to these experiments and may have adapted to higher PI3K/AKT signaling levels during this period.This adaptation could weaken the growth-limiting effects of SHIP1 deletion as compared to the acute inhibitor treatments.To further assess the specificity of our SHIP1 inhibitor, we confirmed that SHIP1-deficient MEC-1 cells were significantly less sensitive to 3AC treatment as compared to control clones (Supplementary Fig. 3h), confirming that the cytotoxic effect is largely due to on-target SHIP1 inhibition.Together, our data clearly shows that SHIP1 expression and activity is required for optimal CLL cell growth and survival. PI3K/AKT hyperactivation promotes oxidative phosphorylation in CLL cells followed by ROS-mediated cell death.To elucidate the mechanism of how hyperactivation impairs CLL progression, we again employed the genetic strategy of PI3K/AKT hyperactivation by forced constitutively active AKT1 expression in MEC-1 cells.After sorting of GFP + myrAKT1 or EVexpressing MEC-1 cells, we conducted RNAseq analysis with subsequent gene set enrichment analysis 49 and found a significant upregulation of genes related to \"oxidative phosphorylation\" upon AKT1 activation in MEC-1 cells (Fig. 5a, b and Supplementary Fig. 4a, Supplementary Data 1).Similar effects were observed in MEC-1 cells upon shRNA-mediated SHIP1 knockdown (Supplementary Fig. 4b).This upregulation was rather surprising, as PI3K/AKT signaling is primarily known to promote the glycolytic metabolic pathway to ensure fast responses to energy demands 50 .To test the metabolic status functionally, we measured the metabolic consequences of AKT1 activation as well as SHIP1 inhibition in CLL cells.Without manipulation, CLL cells primarily rely on mitochondrial oxidative phosphorylation for energy supply 51 .Accordingly, we analyzed mitochondrial functions by determining the oxygen consumption rate (OCR) in MEC-1 cells overexpressing myrAKT1 or empty vector control cells and found higher mitochondrial respiration capacity in the myrAKT1-expressing CLLs (Fig. 5c), which is in line with the enriched \"oxidative phosphorylation\" gene expression signature. Similarly, treatment of MEC-1 cells with the SHIP1 inhibitor 3AC increased OCR levels, both at the baseline and upon respiratory challenge (Fig. 5d).Finally, primary CLL cells also revealed increased mitochondrial capacity upon SHIP1 inhibition, which was less pronounced in healthy donor B cells (Fig. 5e).Glycolytic activity measured by the extracellular acidification rate (ECAR) was not significantly affected by 3AC treatment (Supplementary Fig. 4c).During oxidative phosphorylation, electrons escape from the electron transport chain to induce formation of reactive oxygen species (ROS), including superoxide anions and hydrogen peroxide 52 .To investigate whether the increase in mitochondrial respiration upon SHIP1 inhibition impacts ROS levels in CLL, we measured levels of cellular ROS upon SHIP1 inhibition and indeed observed that the high levels of ROS in primary CLL cells 51 were further increased by 3AC treatment (Fig. 5f).Similarly, in the CLL-derived cell line MEC-1 we found upregulated ROS levels in SHIP1 knockout clones as compared to controls of the MEC-1 line.Treatment of SHIP1 knockout clones with 3AC, however, did not further increase ROS levels (Supplementary Fig. 4d-g), confirming that 3AC-mediated ROS induction is due to on-target SHIP1 inhibition.Importantly, blocking ROS levels derived from oxidative phosphorylation with a mitochondriatargeted antioxidant (mitoTEMPO) significantly reduced the cytotoxicity of SHIP1 inhibition in MEC-1 cells (Fig. 5g) and primary CLL cells (Fig. 5h).Similar results were obtained with the ROS scavenger N-acetyl-cyteine (NAC; Supplementary Fig. 4h,i). These results indicate that PI3K/AKT pathway activation in CLL (by myrAKT1 overexpression, genetic SHIP1 deletion, or by SHIP1 inhibition) promotes the oxidative metabolic pathway in CLL and thereby leads to the formation of toxic ROS levels. SHIP1 inhibition induces lytic cell death with immunogenic features.Given that ROS accumulation plays a key role in mediating different forms of programmed cell death 53,54 , we next investigated which mode of cell death is induced in CLL upon SHIP1 inhibition and tested different inhibitors of cell death pathways in combination with 3AC treatment of primary CLL samples.While pan-caspase-or caspase-8 inhibition had very little or no effect on the cytotoxicity induced by 3AC (Fig. 6a,b), necroptosis inhibition by NEC1s (inhibiting RIP1) or GSK-843 (inhibiting RIP3) did significantly reduce the 3AC-mediated cytotoxicity in primary CLL (Fig. 6c,d).The respective viabilities after the indicated treatments (alone and in combination) are depicted in Supplementary Fig. 4j-m.As necroptosis is an immunogenic form of cell death, we further evaluated whether CLL cells acquired characteristics associated with immunogenic cell death (ICD) 55 upon treatment with the SHIP1 inhibitor 3AC.Translocation of calreticulin to the surface of stressed cells acts as \"eat me\" signal for their removal by phagocytosis 56 .As we expected, calreticulin was exposed to the outer membrane in CLL cells upon SHIP1 inhibition (Fig. 6e).In addition, CLL cells secreted the danger-associated molecular pattern (DAMP) HMGB1 (Fig. 6f) and ATP to the supernatants upon 3AC treatment in a dose-dependent manner (Fig. 6g), which also contributes to the activation of adjacent immune cells.Importantly, in the untreated control CLL cells, no HMGB1 and only low levels of extracellular ATP were detected, despite the high level of spontaneous apoptotic cell death after 48 h in primary CLL cell cultures.This indicates that the release of these immunostimulatory molecules by CLL cells is specifically induced by SHIP1 inhibition rather than secondary, unspecific events during spontaneous cell death.In line with this assumption, HMGB1 release upon SHIP1 inhibition can be partially blocked by inhibiting the MLKL-mediated pore formation using small-molecule inhibition (Fig. 6h).We also observed increased levels of HMGB1 and ATP secreted upon shRNA-mediated SHIP1 knockdown and after myrAKT expression in MEC-1 cells (Fig. 6i,j).These results indicate that SHIP1 inhibition induces a lytic form of cell death with features of necroptosis that triggers the release of immunogenic mediators from CLL cells in vitro.Taken together, we, therefore, conclude that SHIP1 inhibition induces a lytic form of cell death with immunogenic features (summarized in Fig. 6k). ",
"section_name": "SHIP1 inhibition delays CLL progression in vivo.",
"section_num": null
},
{
"section_content": "In this study, we identified a previously unrecognized vulnerability of CLL cells to acute and constitutive activation of the PI3K/AKT signaling pathway.We confirmed that the expression and activity of the inhibitory phosphatase SHIP1 is required to limit PI3K/AKT signaling in CLL cells to prevent excessive ROS production and thereby avoid an immunogenic form of cell death.We, therefore, suggest transient pharmacological targeting of SHIP1 as a therapeutic approach for CLL. When we initiated our investigation of what renders CLL cells specifically vulnerable to AKT1 activation or SHIP1 inhibition, we found that additional activation of AKT1 increases oxidative phosphorylation, thereby triggering ROS-dependent cell death in CLL.In many solid tumors, malignant cells rely on the fast but inefficient generation of ATP via glycolysis to maintain their energy demands in a hypoxic condition with dysfunctional mitochondria.AKT activation has emerged as a central player in this metabolic switch.However, similar to non-transformed, activated B cells, CLL cells circulate in normoxic conditions and primarily rely on mitochondrial oxidative phosphorylation for energy supply, despite their active AKT1 signaling 51,57,58 .Therefore, CLL cells already have high levels of ROS and have evolved mechanisms to cope with this level of oxidative stress, i.e., via upregulation of the antioxidant enzyme hemoxidase 1 51 .Enforced increase of AKT signaling, either via SHIP1 inhibition or genetic activation, however, leads to a further increase in oxidative phosphorylation and to ROS levels toxic for CLL cells.It should be noted that SHIP1 can also have activating roles in cell signaling as PI(3,4)P 2 , the product of SHIP1 dephosphorylating PI(3,4,5)P 3 , can also result in AKT activation 59 .However, our data clearly shows that SHIP1 inhibition results in CLL cell activation, and this activation is critical for SHIP1 inhibitionmediated cytotoxicity.In addition, although genetic PI3K/AKT signaling activation upon introduction of constitutively active mutant AKT1 induces stronger and more persistent signaling compared to SHIP1 inhibition or deletion, we still observed surprisingly similar downstream effects.These effects include the induction of oxidative phosphorylation and ROS accumulation followed by immunogenic cell death.Nevertheless, the induction of cell death was weakened in the genetically manipulated MEC-1 cells as compared to the inhibitor-treated cells, and we speculate that this weakened effect is largely due to adaptation to the higher signaling levels as genetic manipulation takes days and even weeks to successfully create SHIP1 knockdown/knockout lines.In line with the assumption of cellular adaption, genetic knockout of SHIP1 in B cells does not delay the disease development in the TCL1tg mouse model where pre-malignant cells can develop strategies to counteract hypersignaling prior to full transformation 60 . AKT activation has also been linked to promoting cell death in non-hematopoietic cells, particularly if the cells are metabolically challenged 61,62 .Our study demonstrates that AKT activation and increased levels of ROS in CLL cells are critical downstream mediators of SHIP1 inhibition in CLL resulting in a lytic form of cell death.In line with our finding, the induction of necroptotic cell death by TNFα and Caspase inhibition (Z-VAD) in neurons is preceded by the assembly of the critical necroptosis kinases RIP1-RIP3 with activated AKT, and, similar to our observation, pretreatment with small molecule inhibitors of AKT prevented the formation of ROS and necroptosis 63 .Increased ROS levels can be directly detected by RIP1, leading to its activation by autophosphorylation on serine residue 161 64 .This specific phosphorylation then recruits RIP3 and induces the formation of a functional necrosome with pore formation and the release of DAMPs 65 .We observed that RIP1 and RIP3 kinase activity is critical for SHIP1 inhibition-induced cytotoxicity in CLL and this is particularly interesting as we observe features of immunogenic cell death.We further confirmed that CLL cells exhibit key characteristics of immunogenic cell death with the release of immunostimulatory molecules upon SHIP1 inhibition.We hence provide evidence that SHIP1 inhibition promotes an immunogenic form of lytic cell death in CLL, which could be contributing to the treatment efficacy. We had initiated our study based on the hypothesis that strong activation of the PI3K/AKT pathway mimics excessive signaling strength from an autoreactive BCR and that this may be leveraged to trigger negative selection for therapeutic benefit in CLL patients.It is unlikely that autoreactive B cells undergo an immunogenic form of cell death during physiological B-cell selection but most likely undergo classical apoptosis.In T cells, SHIP1 inhibition induces classical apoptosis via Fas/Caspase-8 66 , while our experiments indicate that 3AC-mediated cell death in CLL is largely independent of caspases.One possible explanation for the differential mode of cell death triggered by SHIP1 inhibition in CLL is the upregulated antiapoptotic machinery in CLL, one of the hallmarks of this disease: CLL cells express high ratios of c-FLIP(L) to caspase-8, and thereby prevent caspase-8 activation and apoptosis 67 , which can promote the induction of a necroptotic form of cell death 68 .The sensitivity of CLL cells towards excessive signaling may therefore still resemble negative B-cell selection while the induction of an immunogenic form of cell death upon SHIP1 inhibition specifically occurs in transformed CLL cells. Nevertheless, targeting negative regulators such as SHIP1 also risks promoting unwanted proliferation of other, non-or premalignant cells.Indeed, B-cell-specific knockout of SHIP1 in mice results in the loss of B-cell tolerance and autoimmune manifestations 69 .Similarly, SHIP1 may act as a tumor suppressor in other tumor entities, including AML and T-ALL, and its inhibition may promote proliferation of pre-malignant cells of other origins 70 .However, our therapeutic approach is based on transient SHIP1 inhibition, and potential proliferative signals to other cells are only present for the limited time of treatment.In our short-term treatment schedule in mice, we did not observe any evidence of side effects from SHIP1 inhibition, neither in wt nor in NSG mice.Similarly, other groups reported extended survival of mice challenged with multiple myeloma 71 with no apparent toxicity upon SHIP1 inhibitor in vivo treatment, even upon continuous treatment with 3AC in immunocompetent mice 44,72 .We hypothesize that some of the effects of SHIP1 inhibition on other, non-CLL cells may also increase treatment efficacy.Recent studies have demonstrated that SHIP1 inhibition in different tumor models can increase the anti-tumor NK-and T-cell responses 73 .This is particularly promising in the context of CLL where patients suffer from drastic immunosuppression leading to fatal infections causing up to 30-50% of CLL-related mortalities 74 .We, therefore, suggest that both cell autonomous and nonautonomous mechanisms can contribute to the therapeutic efficacy of SHIP1 inhibition in CLL.We speculate that transient SHIP1 inhibition in repetitive cycles can cause lytic cell death with immunogenic features in the malignant cells and simultaneously enhance the immunoresponse by directly acting on NK-and T cells to restore effective immune responses and potential anti-tumor immunity in CLL. Taken together, our results show that CLL cells rely on a delicate coordination between the cellular signaling pathways regulating metabolic processes for their cellular growth and depend on intermediate signaling.We show that perturbation of negative regulation of the PI3K/AKT signaling, even if counterintuitive, induces a ROS-mediated lytic and immunogenic form of cell death in CLL.We, therefore, propose transient inhibition of SHIP1 as an unexpected concept for CLL therapy, either alternating with kinase inhibition to potentially enhance the effect of both solo treatments or to treat the rising cases of kinase inhibitor-resistant disease. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Human subjects.Primary CLL samples were obtained from the peripheral blood of patients at the National Center for Tumor Diseases, Heidelberg, Germany (CLL1-CLL30, Supplementary Table 1).Data for IgV H status, SHIP1 expression was obtained by RNA Sequencing 39 .Clinical data and gene expression were analyzed by Junyan Lu (EMBL Heidelberg).In addition, Klinikum München Schwabing (Clemens Wendtner; CLL31-CLL36) and Tumor Therapy Center of Klinikum rechts der Isar (Christian Bogner, CLL37-CLL44) provided peripheral blood of CLL patients for xenotransplantation approaches and in vitro experiments.The local ethics committee of the Faculty of Medicine, Technical University Munich, approved patient and healthy donor sampling and all presented experiments.All participants (CLL patients and healthy donors) gave informed consent.All patients were treatment naïve or off CLL therapy for at least 3 months.Healthy donor-derived blood samples (age-matched) were received from the \"Bayerisches rotes Kreuz\" (Munich, Germany). Cell lines.Chronic B-cell leukemia-derived cell lines MEC-1 (RRID: CVCL_1870), EHEB (RRID: CVCL_1194), and lymphoma lines SUDHL6 (RRID: CVCL_2206), BJAB (RRID: CVCL_5711), and Bal17 (RRID: CVCL_9474) were purchased from DSMZ (Braunschweig, Germany).MEC-1 (Slc7a) Eco cells were generated by transduction with pLenti6/UbC/mSlc7a1, a gift from Shinya Yamanaka (Addgene plasmid # 17224; RRID: Addgene_17224) and MEC-1 Luciferase positive cells were generated by the introduction of pCL6-Luc-GFP into MEC-1 Eco cells by retroviral transduction. Quantitative real-time PCR (qPCR).RNA was isolated from sorted bone marrow-or spinal cord-residing MEC-1 cells by using RNeasy Plus Micro Kit (QIA-GEN) according to the manufacturer's instructions.RNA concentration of the samples was determined by NanoDrop.RNA was reverse transcribed using the qScript cDNA SuperMix (Quantabio) according to the manufacturer's instructions with 20-1000 ng of total RNA.The generated cDNA was used in triplicates for RT-PCR reactions, with primers that span exon-exon boundaries to ensure cDNAspecific amplification.The qPCR Kit Takyon™ No ROX SYBR® 2X MasterMix dTTP Blue (Eurogentec) was used to perform RT-PCR.Gene expression was normalized to the housekeeping gene GAPDH.The reaction was performed in a Light Cycler 480 II (Roche).All primer sequences are listed in Supplementary Table 2. myrAKT1 in MEC-1.pMIG-emtpy vector (EV) and pMIG-myrAKT1 were kindly provided by Hassan Jumaa 32 and by retroviral transduction introduced into MEC-1 Eco cells.GFP content was followed over time by flow cytometric analysis and DAPI exclusion.The expression of active AKT was confirmed by western blot. Mice.Mice were housed according to the guidelines specified in the EU Directive 2010/63 with a light-dark rhythm of 12 h each with twilight phase, air condition (at 20-24 °C temperature), and a humidity of 45-60%.The AKT1 E17K cDNA, carrying a point mutation at nucleotide position 49 (G to A) in the human AKT1 gene, was cloned into the ubiquitously expressed ROSA26 vector, preceded by a loxP-flanked transcriptional and translational STOP cassette.Electroporation of 129J/Ola embryonic stem cells and generation of chimeric mice were performed by Polygene, Switzerland.Successful recombination of embryonic stem cell clones was evaluated by Southern Blot analysis of genomic DNA digested with XbaI 36 .Germline transmission was confirmed by PCRs specific for the targeted locus.All primers used are listed in Supplementary Table 2.The bicistronic expression of AKT1 E17K together with eGFP preceded by an internal ribosomal entry site (IRES) sequence allowed fluorescence monitoring of AKT1 E17K expressing cells.Blastocyst injection of the clones and subsequent chimera breeding resulted in AKT1 E17K Fig. 5 Mechanistic analysis of AKT1 activation/SHIP1 inhibition in CLL. a Heatmap analysis of differentially regulated genes associated with oxidative phosphorylation in MEC-1 cells upon transduction with pMIG EV (left) or pMIG-myrAKT1 (right), representative for two independent experiments.Color scale indicates the Z score (red = up; blue = down).b Gene set enrichment analysis (GSEA) results for the association with \"oxidative phosphorylation\" is shown upon myrAKT expression, Enrichment Score (ES) 0.65666306; Normalized Enrichment Score (NES) 2.847461; Nominal p-value < 0.001; FDR qvalue < 0.001; FWER p-value < 0.001; representative for two independent experiments.c Oxygen consumption rate (OCR) was measured in GFP-sorted MEC-1 cells upon transduction with pMIG EV or pMIG-myrAKT1, pooled analysis of three independent experiments.Data are presented as mean values ± SEM (left) and as individual values and mean values ± SD in the bar graph (right).Statistical significance was assessed by a two-tailed unpaired Student's ttest.d OCR was measured in MEC-1 cells treated for 1 h with SHIP1 inhibitor (SHIP1i; 5 μM 3AC; clear squares) or control (black squares), pooled analysis of seven independent experiments.Data are presented as mean values ± SEM over time (left) and as individual values and mean values ± SD in the bar graph (right).Right: Statistical significance was assessed using mean values of three independent experiments by a two-tailed unpaired Student's t-test.e OCR was measured in a healthy donor (HD) B cells (n = 6; circles) and primary CLL cells (n = 6, squares) upon 1 h treatment with SHIP1i (5 μM 3AC; clear) or control (filled); pooled data from two independent experiments.Data are presented as mean values ± SEM (left) and as individual values and mean values ± SD in the bar graph (right).Statistical significance was assessed by a two-tailed unpaired Student's t-test.f Quantification of MFI upon ROS analysis using the CellROX orange dye in primary CLL cells untreated, or treated for 4 h with 5 μM 3AC is shown (n = 8).Data are presented as individual values per CLL donor.Statistical significance was assessed by a two-tailed paired Student's t-test.g MEC-1 cells were treated with MitoTEMPO (MT; filled squares), SHIP1i (3AC; clear squares) or the combination (clear circles) for 24 h treatment, measured in five independent experiments; the specific cell death was determined as described in Fig. 2e.Data are presented as individual values and mean values ± SD.Statistical significance was assessed by a twotailed unpaired Student's t-test.h Primary CLL samples (n = 3) were treated with MT (filled squares), SHIP1i (3AC; clear squares), or the combination (clear circles) for 24 h; the specific cell death was determined as described in Fig. 2e.Data are presented as individual values and mean values ± SD.Statistical significance was assessed by a two-tailed unpaired Student's t-test.Significance values are depicted in the graph; (n.s.) not significant.Source data are provided as a Source Data file.transgenic mice that were then crossed to Mb1-CreER T2 mice 37 and the TCL1tg mouse model 33 . For xenotransplant experiments, we used NSG mice (NOD.Cg-Prkdcscid Il2rgtm1WjI/SzJ), purchased from Charles River or Janvier laboratory as recipients for the human MEC-1 CLL-like cell line (purchased from DSMZ, Braunschweig, Germany) or primary CLL patient samples.For treatment of murine CLL in vivo, we transplanted 2 × 10e7 splenocytes of aged TCL1tg mice 33 into C57BL/6N (Janvier Labs) wt immunocompetent mice and waited for detection of CLL or full engraftment and disease progression.Treatment schedules are depicted in Supplementary Fig. 3.All animal experiments were carried out in accordance with the guidelines of the Federation of European Laboratory Animal Science Association (FELASA) and followed the legal approval of the Government of Upper Bavaria (Regierung von Oberbayern). AKT1 hyperactivation in the TCL1tg model via Cre-mediated recombination in vitro and in vivo.Isolated splenocytes of AKT1 E17K × TCL1tg or TCL1wt littermates were treated with 500 ng/ml 4-OHT for 48 h or with TAT-Cre (Excellgen) diluted in Opti-MEM at a final concentration of 2 μM for 1 h at 37 °C, 5% CO 2 , and 95% humidity.Afterward, cells were washed twice in culture media.The induction of the AKT1 transgene was analyzed by flow cytometric measurement of GFP percentage and DAPI exclusion.To activate AKT1 E17K in vivo, mice transplanted with above described leukemic triple transgenic splenocytes were induced to express AKT1 E17K by providing tamoxifen-enriched chow (400 mg tamoxifen citrate kg -1 chow; CreActive TAM400, LASvendi, Soest, Germany) or control chow for 4 weeks upon transplantation to activate CreER T2 .CLL content and GFP expression in the peripheral blood were followed by flow cytometry and after 2-month mice were sacrificed, organs harvested, and analyzed by flow cytometry.Cell isolations.Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by density gradient centrifugation using Ficoll-Paque (GE Healthcare, Chicago, IL, USA).CD19 + B cells, including MEC-1 cells, were purified by magnetic-activated cell sorting (MACS) using human B-cell isolation kit II or human CD19 MicroBeads (Miltenyi, Bergisch-Gladbach, Germany).Purification of primary CLL cells (CD19 + CD5 + ) and MEC-1 cells (GFP, CD19 + ) was performed using fluorescence-activated cell sorting (FACS) (BD Aria II, BD Bioscience, Franklin Lakes, NJ, USA).All antibodies are listed in Supplementary Table 3. Peripheral blood, spleen, and axillary lymph nodes were harvested per mouse. Organs were meshed through a 70 μm cell strainer in PBS buffer and erythrocytes were lysed using G-DEXTMIIb RBC Lysis Buffer (Intron Biotechnologies). Western blot.Whole-cell lysates for protein analysis were prepared in CHAPS buffer, supplemented with phosphatase inhibitors (50 mM NaF, 0.1 mM Na 3 VO 4 , and protease inhibitors in resolved EDTA-free Protease Inhibitor Cocktail Tablets, Roche Diagnostics) for 15 min on ice using standard methods.BCA Protein Assay Kit (ThermoFisher Scientific) was applied for protein concentration determination.4-12% gradient gels from Invitrogen, NuPage were applied according to manufacturer's instructions.To analyze the release of HMGB1, supernatants were collected upon 48 h 3AC treatment by centrifugation (400 × g, 5 min, 4 °C) and Laemmli buffer was added, according to standard protocols.Samples were loaded on 10% Tris/Bis gels and run in SDS running buffer for 1.5 h at 125 V.The separated proteins were transferred onto a nitrocellulose membrane (Amersham Protran, GE Healthcare) by wet-blot electrophoresis for 2 h at 300 mA.Antibodies are listed in Supplementary Table 3. Flow cytometry.Cells were stained with fluorochrome-labeled antibodies according to manufacturer information (listed in Supplementary 75 were treated with 3AC starting at day 1 after transplantation for 4-8 daily doses.Peripheral blood (PB) analysis determined the endpoint and organs were harvested when <10% human CLL cells were detectable in the PB.In the murine TCL1tg model, 3AC treatment was initiated when PB of transplanted mice confirmed a CLL positive population <5% or when CLL content was on average 50% in the peripheral blood.Mice were treated daily, in different treatment cycles as described in Supplementary Fig. 3. Organs were harvested at experimental termination and analyzed for CLL content by flow cytometry.Samples for histology were fixed in 4% PFA. Histology.Murine spleens were fixed in 10% neutral buffered formalin (for 48 h) and then dehydrated and embedded in Paraffine (Leica ASP 300S) according to routine methods.To detect and analyze infiltration with neoplastic cells, blocks were cut (2 μm thickness) and stained with Hematoxylin-Eosin or anti-human CD20cy antibody (Agilent, clone L26, 1:2000).CD20 IHC was performed on a Leica BondRxm using a Polymer Refine detection kit.All slides were scanned with a Leica AT2 scanning system with ×40 magnification.HE stainings and IHCs were evaluated by a board-certified veterinary pathologist and the amount of CD20 positive neoplastic cells in the spleen was analyzed by using the Aperio Positive-PixelCount v9.The number of positive pixels was calculated per mm 2 and the results between the groups (3AC-treated vs. control) were visualized and statistics (Mann-Whitney U test) were calculated with IBM SPSS statistics 25. Bioimaging.MEC-1 Eco cells were transduced with firefly luciferase retrovirus and selected with blasticidin for bioluminescence imaging 14 . 1 h after transplantation of MEC-1 Luc cells into NSG mice, 2 mg Luciferin were i.p. administered per mouse.After a 10 min integration time, luminescence in mice was captured using the IVIS Lumina imaging system (Perkin Elmer) and exposed for 10 s, 1 min, and 5 min.The Living Image Software (Perkin Elmer) was used for analysis. SHIP1 activity assay.Whole-cell lysates of 1 × 10 8 primary cells of CLL patients (n = 4) and isolated B cells of healthy donor-derived samples (n = 5) were prepared using NP40 buffer for the SHIP1 inositol phosphatase activity assay 29 .H1299 cells that lack endogenous SHIP1 expression were used to lentivirally overexpress wt SHIP1 or the AML-derived R673Q variant of SHIP1 and served as a positive and negative control, respectively.1 mg of each lysate was used for immunoprecipitation of SHIP1 using a mouse monoclonal antibody (SHIP1 P1C1, Santa Cruz Biotechnology), which had been coupled to G sepharose beads.For the assay, the buffer was changed from NP40 buffer to phosphatase assay buffer and the beads containing immunoprecipitated SHIP1 were separated in four parts per sample.Three parts were used for the three assay replicates and the remaining part for western blot analysis to evaluate the amount of immunoprecipitated SHIP1.For the assay, a commercially available phosphate assay kit (Bioassay systems) was used with 20 μM f.c.ci8-Phosphatidylinositol-3,4,5-trisphosphate as substrate in a final volume of 400 μl.The sample was preincubated for 5 min at 37 °C after which the ) and the specific cell death was determined as described in Fig. 2e.For the combination treatment, the viability of the single treatments of the cell death inhibitors was used for determining the baseline cell death to calculate the specific cell death.Viability is depicted in Supplementary Fig. 4j-m substrate was added.90 μl aliquots were taken after 5, 20, 60, and 120 s, and the reaction was stopped by the addition of 31.5 μl of 0.1 M EDTA.The amount of released phosphate was evaluated photometrically as instructed in the assay kit.To determine the enzyme activity, the initial change in nmol phosphate (5-20 s)/min was calculated. Cell stimulation and inhibitor treatment.Murine TCL1tg splenocytes (>70% CLL), primary patient-derived peripheral blood CLL cells (>70% CLL) and healthy peripheral blood MACS-isolated B cells were seeded at 200,000 cells/well in 96-well u-bottom plates or 500,000 cells in 24-well plates and treated with 3AC (Echelon Bioscience) (or K118 (Echelon Biosciences) when indicated) up to 48 h.Viability was analyzed by flow cytometry.In combinatorial treatment experiments with the inhibitors GSK-843 (Selleckchem; 1 μM), Z-IETD (BD Bioscience; 10 μM), Emricasan (Selleckchem; 5 μM), AZD-5363 (Toric Bioscience; 5 μM), NEC1s (BioVision; 30 μM), NSA (Cayman Chemical; 2μM) cells were pre-treated for 1 h, followed by addition of 5 μM 3AC for 48 h.Cell lines (MEC-1, EHEB, Bal17, SUDHL6, BAJB) were synchronized by cell count and resuspended at 1 × 10 6 cells/ ml in a fresh culture medium 1 day prior to treatment.Cells were then treated with 3AC at 100,000 cells/well in 96-well u-bottom plates or 500,000 cells/well in 24-well plates. ATP measurement.To measure the release of ATP, patient-derived CLL cells were treated for 4 h with 3AC as described and supernatants were analyzed with the ATP Enliten Kit (Promega), according to manufacturer's instructions.Similarly, supernatants of puromycin-selected (shRNA expressing, d6) or GFP-sorted (EV, myrAKT expressing, d5) MEC-1 cells were collected and analyzed with the Cell Titer Glo Kit (Promega) according to the manufacturer's instructions.For detection of luminescence the GloMax® Discover platereader was used (Promega). Specific cell death.DAPI negative cells were defined as viable cells via flow cytometry.Percentage of specific cell death was calculated by this formula: specific cell death (%) = 100 × (% dead cells -% baseline dead cells)/(100 -% baseline dead cells). Genetic knockdown.shRNA SHIP1 knockdown1 (KD1) and shRNA SHIP1 knockdown2 (KD2) as well as scramble (scr) were purchased from Sigma Aldrich and subcloned into pLKO.1.For the generation of SHIP1 KD MEC-1 cell lines, MEC-1 Eco and MEC-1 Eco/Luc cells were transduced with scramble or 2 constructs of SHIP1-targeting shRNA (KD1, KD2) concentrated lentivirus (pCMV delta R8.2: Addgene #12263, RRID: Addgene_12263), Phit123: kindly provided by Markus Müschen).5 days after spin infection, Puromycin selection was initiated with 1 μg/ml for 3 days.Cell viability before and after Puromycin selection was assessed by flow cytometric DAPI exclusion and by total cell count.SHIP1 knockdown was confirmed by western blotting.1 × 10 6 shSHIP1 KD1, KD2 MEC-1 cells, and shScramble control-containing MEC-1 cells were transplanted into NSG mice and CLL engraftment and progression was followed by bioimaging as described above.Bone marrow and spinal cord cells were isolated from sacrificed mice and MEC-1 (GFP + , CD19 + ) cells were FACS sorted and SHIP1 expression levels were analyzed by qPCR. Genetic knockout.To generate SHIP1 knockout (KO) CLL cells, MEC-1 Eco cells were transduced with INPP5D-targeting guide RNAs (5 different sgRNAs, listed in Supplementary Table 2) and pMIG-Cas9 containing retrovirus 14 .After 48 h from transduction cells were selected with 1 μg/ml of puromycin for 3 days.The selected cells were seeded as single colonies in 96-well plates by FACS sorting.After 3-4 weeks of culture, cells derived from each colony were used to assess SHIP1 knockout by western blotting and genomic sequencing of the sgRNA target region (Amplification and sequencing primer are listed in Supplementary Table 2). 2 × 10 6 SHIP1 KO (3 independent clones) and SHIP1 wild-type (2 independent clones) MEC-1 cells were transplanted into NSG mice.The mice were sacrificed upon clear signs of disease, comprising neurological symptoms and weight loss.Prior to transplantation, all cells were kept overnight at 1 × 10 6 /ml in fresh media, and equal viability was confirmed prior to injection. Competition assay.The growth behavior of MEC-1 SHIP1 KO cells was analyzed by competition assays.500,000 GFP-positive knockout cells were mixed with equal amounts of MEC-1 wild-type, GFP-negative cells.The percentage of GFPexpressing cells was followed by flow cytometric analysis over time. RNA preparations/RNASeq.For bulk 3′-sequencing of poly(A)-RNA (RNASeq), viable GFP + MEC-1 cells on day 7 post-transduction with pMIG-empty (EV) or pMIG-myrAKT1 were sorted and RNA was extracted from whole-cell lysates via RNeasy Mini Kit (Qiagen, Hilden, Germany).Barcoded cDNA of each sample was generated with a Maxima RT polymerase (ThermoFisher) using oligo-dT primer containing barcodes, unique molecular identifiers (UMIs), and an adapter.5′ ends of the cDNAs were extended by a template switch oligo (TSO) and after pooling of all samples, full-length cDNA was amplified with primers binding to the TSO-site and the adapter 76 .cDNA was fragmented and TruSeq-Adapters ligated with the NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® (NEB) and 3′-endfragments were finally amplified using primers with Illumina P5 and P7 overhangs. In comparison to Parekh et al., the P5 and P7 sites were exchanged to allow sequencing of the cDNA in read1 and barcodes and UMIs in read2 to achieve a better cluster recognition.The library was sequenced on a NextSeq 500 (Illumina) with 75 cycles for the cDNA in read1 and 16 cycles for the barcodes and UMIs in read2.For analysis, gencode gene annotations v28 and the human reference genome GRCh38 were derived from the Gencode homepage (EMBL-EBI).Drop-Seq tools v1.12 77 were used for mapping raw sequencing data to the reference genome.Shown experiments (myrAKT1 overexpression and Ship1 knockdown with the respective controls) were separately processed.The resulting UMI filtered count matrices were imported into R v3.4.4.CPM (counts per million) values were calculated for the rawdata and genes having a mean CPM value less than 1 were removed from the dataset.Prior differential expression analysis with DESeq2 v1.18.1 78 , dispersion of the data was estimated with a parametric fit using an univariate model where treatment was specified as an independent variable.The Wald test was used for determining differentially regulated genes between treaetments within each individual patient and shrunken log2-fold changes were calculated afterwards.A gene was determined as differentially regulated if the absolute apeglm shrunken log2-fold change was at least 1 and the adjusted p-value was below 0.01.GSEA v4.0.3 49 was performed in the weighted Preranked mode, where either the Wald test-statistic or the apeglm shrunken fold change was used as ranking metric.All genes tested for differential expression were used for GSEA analysis, with genesets from MsigDB v7.1 79 where used for testing.A pathway was considered to be significantly associated with treatment if the FDR value was below 0.05.Rlog transformation of the data was performed for visualization and further downstream analysis.Genes of Oxphos pathway contributing most to the NES score of the GSEA are displayed as Heatmap.Heatmap shows z-transformed expression data.Raw sequencing data are available from the European Nucleotide Archive under the accession number PRJEB38070. Oxidative stress.Cellular and mitochondrial ROS levels were determined by flow cytometry using CellROX ® Orange (ThermoFisher Scientific).To analyze the impact of ROS in therapy-induced cell death, CLL cells were co-treated with 3AC in presence of ROS scavengers MitoTEMPO (10 μM) (Merck) or NAC (2 mM) (Nacetyl-cysteine) (Sigma Aldrich) for 24-48 h. Extracellular flux analysis.Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XFe96 Flux Analyzer with the XF Cell Mito Stress Test Kit and XF Glycolysis Stress Test Kit (Agilent) according to the manufacturer's instructions.All compounds and materials were obtained from Agilent. Statistical analysis.Statistical significance was analyzed with paired or unpaired two-tailed Student's t-test, ordinary one-way ANOVA or log-rank (Mantel-Cox), using Prism Version 7.0, Graphpad Software Inc., as indicated. Reporting summary.Further information on experimental design is available in the Nature Research Reporting Summary linked to this paper. ",
"section_name": "Methods",
"section_num": null
}
] |
[
{
"section_content": "We thank Junyan Lu for bioinformatics analysis of the RNAseq data from primary CLL samples and clinical association, Paul König for support with CRISPR/Cas9 targeting, Elias Hobeika and Michael Reth for providing the Mb1-CreER T2 mouse line, Hassan Jumaa for providing the myrAKT1 plasmid, Ulrike Höckendorf and Philipp Jost for cell death inhibitors and helpful discussions on immunogenic cell death, Marc Schmidt-Supprian for helpful discussions, Dirk Wohlleber and Percy Knolle for access to the IVIS Bioimager, Christian Bogner and Simon Heidegger for providing primary CLL samples from Klinikum rechts der Iar.This work was funded by the German Cancer Aid (Deutsche Krebshilfe, Max Eder Grant to M.B.), the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) -Project-ID 360372040 -SFB 1335/P02 awarded to M.B., P01 to J.R. and Z01 to K.S., the international doctoral program \"i-Target\" funded by the Elite Network of Bavaria (to J.R.) and the Krebsliga (to T.Z.). ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Open Access funding enabled and organized by Projekt DEAL. ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "All codes used for RNAseq analysis are available upon reasonable request.Source data are provided with this paper. ",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "All codes used for RNAseq analysis are available upon reasonable request.Source data are provided with this paper. ",
"section_name": "Code availability",
"section_num": null
},
{
"section_content": "The RNA sequencing data referenced during the study are available in a public repository from the European Nucleotide Archive (ENA) under the accession code PRJEB38070.INPP5D mutations in CLL can be found on cBioPortal, freely available.All the other data supporting the findings of this study are available within the article and its Supplementary Information files and from the corresponding author upon reasonable request.Source data are provided with this paper. V.E.: Data acquisition, methodology, analysis, and manuscript preparation; M.S.: data acquisition, methodology, analysis, and manuscript preparation; L.B.: performed part of the in vivo experiments; T.N.: technical support with data acquisition; L.P.: performed SHIP1 western blot analysis; T.E.: performed the bioinformatical analysis of the RNA sequencing data; I.R.: provided guidance and reagents; N.N.: performed the SHIP1 activity assay, M.J.: performed the SHIP1 activity assay, manuscript preparation; S.W.: generation of the AKT1 E17K transgenic mouse model; T.Z.: provided primary CLL samples and helpful discussion; C.W.: provided primary CLL samples and helpful discussion; K.M.: supported the IVIS bioimaging measurements.K.S.: performed and analyzed histology sections for human CLL load.R.R.: performed the bioinformatical analysis of the RNA sequencing data.M.M.: provided guidance and reagents, manuscript preparation; J.R.: generation of the AKT1 E17K transgenic mouse model, provided guidance, helpful discussion, and supported manuscript preparation; M.B.: Conceptualization, project administration, funding acquisition, and manuscript preparation.All authors discussed the results and contributed to the manuscript. The authors declare no competing interests. The online version contains supplementary material available at https://doi.org/10.1038/s41467-021-23752-2. Correspondence and requests for materials should be addressed to M.B. Peer review information Nature Communications thanks William Kerr, Paul Liu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Reprints and permission information is available at http://www.nature.com/reprintsPublisher'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": "The RNA sequencing data referenced during the study are available in a public repository from the European Nucleotide Archive (ENA) under the accession code PRJEB38070.INPP5D mutations in CLL can be found on cBioPortal, freely available.All the other data supporting the findings of this study are available within the article and its Supplementary Information files and from the corresponding author upon reasonable request.Source data are provided with this paper. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "V.E.: Data acquisition, methodology, analysis, and manuscript preparation; M.S.: data acquisition, methodology, analysis, and manuscript preparation; L.B.: performed part of the in vivo experiments; T.N.: technical support with data acquisition; L.P.: performed SHIP1 western blot analysis; T.E.: performed the bioinformatical analysis of the RNA sequencing data; I.R.: provided guidance and reagents; N.N.: performed the SHIP1 activity assay, M.J.: performed the SHIP1 activity assay, manuscript preparation; S.W.: generation of the AKT1 E17K transgenic mouse model; T.Z.: provided primary CLL samples and helpful discussion; C.W.: provided primary CLL samples and helpful discussion; K.M.: supported the IVIS bioimaging measurements.K.S.: performed and analyzed histology sections for human CLL load.R.R.: performed the bioinformatical analysis of the RNA sequencing data.M.M.: provided guidance and reagents, manuscript preparation; J.R.: generation of the AKT1 E17K transgenic mouse model, provided guidance, helpful discussion, and supported manuscript preparation; M.B.: Conceptualization, project administration, funding acquisition, and manuscript preparation.All authors discussed the results and contributed to 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": "",
"section_name": "Additional information",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https://doi.org/10.1038/s41467-021-23752-2. Correspondence and requests for materials should be addressed to M.B. Peer review information Nature Communications thanks William Kerr, Paul Liu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Reprints and permission information is available at http://www.nature.com/reprintsPublisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ",
"section_name": "Supplementary information",
"section_num": null
}
] |
10.1186/s13045-019-0802-x
|
Genetic dynamics in untreated CLL patients with either stable or progressive disease: a longitudinal study
|
<jats:title>Abstract</jats:title><jats:p>Clonal evolution of chronic lymphocytic leukemia (CLL) often follows chemotherapy and is associated with adverse outcome, but also occurs in untreated patients, in which case its predictive role is debated. We investigated whether the selection and expansion of CLL clone(s) precede an aggressive disease shift. We found that clonal evolution occurs in all CLL patients, irrespective of the clinical outcome, but is faster during disease progression. In particular, changes in the frequency of nucleotide variants (NVs) in specific CLL-related genes may represent an indicator of poor clinical outcome.</jats:p>
|
[
{
"section_content": "In chronic lymphocytic leukemia (CLL), the clonal expansion acquired relevance with the NGS era, which allowed its use for clinical monitoring.Research was mainly performed on large CLL cohorts sampled before and after therapy [1] and only a few studies investigated clonal evolution longitudinally in stable versus progressive untreated patients [2][3][4].The key results indicate expansion of specific clones upon therapy and heterogeneity of mutated genes among patients, but the extent to which the genetic dynamics differs between stable and progressive untreated CLLs is still controversial. To address this point, we used a CLL cohort including untreated sequential samples from patients with either progressive (P-CLL) or stable (S-CLL) disease.Patients' features are in Additional file 1: Table S1.At each time point, the diagnosis of stable or progressive CLL was established by the clinicians according to the criteria defined during the International Workshop on Chronic Lymphocytic Leukemia [5].Using genome-wide copy number variation (CNV) analysis, we investigated copy number fluctuations in 11 stable CLLs (S-CLLs) and 15 progressive CLLs (P-CLLs).Data were processed using the Rawcopy package [6], and paired segments were defined for each patient (Additional file 2: Figure S1).Since the percentage of CLL cells (f) in PBMCs was not always known, analyses were performed varying f from 1 to 100%.To define aberrant loci, we used two sets of thresholds on log ratio (LogR) value, depending on f and on copy number (k) in CLL cells (Additional file 2: Figure S2).We did not find significant differences in percentages of aberrant loci between S-CLLs and P-CLLs (Fig. 1a), but the rate of change (or slope), reflecting the rate of aberrant clones evolving over time, was significantly higher in P-CLLs (p ≤ 0.05, Mann-Whitney U test) (Fig. 1b).Thus, S-CLLs and P-CLLs seemed to have the same probability of acquiring or losing clones, but this phenomenon was faster in P-CLLs.The results were validated on 6 S-CLLs and 5 P-CLLs with known percentage of CLL cells in PBMCs (Additional file 2: Figure S3-S4), suggesting that tracking copy number changes does not mandatorily require knowledge of cancer cell percentage. To identify genetic events associated with faster clonal expansion, we characterized the CLL-specific genetic features of our cohort.Analyses by qPCR of three chromosomal abnormalities of prognostic value, del (11q), tri (12), and del (17p) [7], did not reveal significant differences between S-CLLs and P-CLLs (Additional file 2: Figure S5).Subsequently, we characterized 11 S-CLLs and 17 P-CLLs for point mutations or indels in regions of 27 genes reported as mutated in CLL (Additional file 3: Table S2).We did not register any significant difference between S-CLLs and P-CLLs with regard to frequency and number of nucleotide variants (NVs) (data not shown).Next, we focused only on NVs with variant allele frequencies (VAF) changing more than 20% between longitudinal samples (dynamic NV: dNVs, synonymous or non-synonymous).We detected on average 1.18 and 3.35 dNVs per sample in S-CLLs and P-CLLs, respectively (Additional file 4: Table S3).P-CLLs showed higher gains/increases of dNVs (p = 0.0008, Fisher's test) (Fig. 2a).Patients with dNV > 1 had shorter treatment-free survival (TFS), considering as starting point the date at first sampling (p = 0.0029) or at diagnosis (p = 0.0004, log rank test) (Fig. 2b and Additional file 2: Figure S6).A dNV > 1 was also associated with poor prognostic factors, including unmutated IGVH and trisomy 12 (p = 0.0461 and p = 0.0407, respectively, Fisher's test) (Additional file 5: Table S4).Patients with unmutated IGVH showed Fig. 2 Mutational status of CLL samples.a Next-generation sequencing of 27 CLL-associated genes in 11 and 17 patients with stable and progressive disease, respectively.Sequence variants were identified using Torrent Suite 3.4 and Variant Caller plugin 3.4.4.NVs with a coverage < 100 were not considered; NVs with a mutation frequency < 5% were not considered; NVs residing in homopolymer DNA sequences (≥ 4 nucleotides) were also not considered.Each square report either gain or loss of mutation or change in mutation frequency (> 20%) between FTP (left side) and LTP (right side); the mutation frequency of dynamic nucleotide variants (dNVs) was reported in Additional file 4: Table S3.Fisher's exact tests were used to compare groups.Statistical tests were two-sided, and significance was defined as p < 0.050.b Kaplan-Meier curve of treatment-free survival in CLL patients dichotomized based on the number of NVs that change more than 20% between FTP and LTP (dNVs) (upper panel) and on the mutational status of the poor prognostic factor IGVH (lower panel).The median of dNVs changed across all the samples was used as cut-off.Time to treatment was calculated from the first sampling; the last follow-up was considered for patients, which did not undergo treatment.The Log-rank test was used to test for significance.c Mutation frequency of dNVs in stable and progressive CLL groups, and in mutated or unmutated IGVH CLL groups; data are reported as median and interquartile range (box); whiskers range from min to max.Mann-Whitney test was used to compare groups; **denotes a p value ≤ 0.01 shorter TFS, supporting the reliability of our cohort (Fig. 2b).Finally, we found that in P-CLLs the average of dNV frequencies was higher in the first sample (p = 0.0074, Mann-Whitney U test), where it was not associated with IGVH mutational status (Fig. 2c).These findings suggest that dNVs could have an exploitable clinical relevance.However, since dNVs include synonymous/non-synonymous mutations and NVs in non-coding regions, we cannot speculate on the molecular role of the targeted genes most frequently mutated, such as ITPKB and NOTCH1 (Fig. 2a).Indeed, these dNVs were only used here to track genetic evolution. In conclusion, differently from previous studies, we calculated VAFs on PBMCs, demonstrating that this is reliable to track CLL evolution.In fact, an increase of a single VAF over time indicates expansion of the clone carrying that NV, regardless of variation in cancer cell fraction.Overall, our study points to a higher genetic dynamics in P-CLLs and suggests that monitoring VAFs of a specific gene panel in PBMCs from sequential samples of a CLL patient may predict disease progression. ",
"section_name": "To the Editor",
"section_num": null
}
] |
[
{
"section_content": "We thank Ms. Lia De Amicis for the administrative work.We thank Valerie Matarese for the manuscript editing and Prof. Carlo Maria Croce for providing DNA samples. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This study is supported by the Italian Association for Cancer Research (AIRC) with Start Up grant 2010 (10054) to RV and partially by the Italian Association for Cancer Research (AIRC IG-17063) to SV. RV was supported by her own Marie Curie Career Integration Grant (GA-2011-303735). ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "Availability of data and materials DNA CNVs and mutational data are freely available to ArrayExpress database (accession number E-MTAB-8020) and European Nucleotide Archive database (accession number ERP115524).All the other raw data are freely available at the code-hosting platform GitHub (https://github.com/VeroneseVisoneLabs/Genetic-dynamics-in-untreated-CLL-patients-with-either-stable-or-progressivedisease-a-longitudinal). ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Supplementary information accompanies this paper at https://doi.org/10.1186/s13045-019-0802-x. Additional file 1: Table S1 Molecular and clinical features of all patients included in the study. Additional file 3: Table S2 HaloPlex SureDesign Report. Additional file 4: Table S3 Genetic variants represented in Fig. 2. Additional file 5: Table S4.Characteristics of patients having few (dNV≤1) versus many (dNV>1) nucleotide variants. Additional file 6: Table S6.SNPs and mutations described by dbSNP and COSMIC databases, respectively, and localized in the regions analyzed by Haloplex SureDesign. Additional file 7: Table S7.SNPs and mutations detected by Haloplex SureDesign Panel. The institutional review board of the University of California, San Diego (171884CX), and of the Fondazione Policlinico Agostino Gemelli (P/948/CE/ 2011) approved the research protocol.Samples were provided upon written informed consent. 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": "Supplementary information accompanies this paper at https://doi.org/10.1186/s13045-019-0802-x. Additional file 1: Table S1 Molecular and clinical features of all patients included in the study. ",
"section_name": "Supplementary information",
"section_num": null
},
{
"section_content": "Additional file 3: Table S2 HaloPlex SureDesign Report. Additional file 4: Table S3 Genetic variants represented in Fig. 2. Additional file 5: Table S4.Characteristics of patients having few (dNV≤1) versus many (dNV>1) nucleotide variants. Additional file 6: Table S6.SNPs and mutations described by dbSNP and COSMIC databases, respectively, and localized in the regions analyzed by Haloplex SureDesign. Additional file 7: Table S7.SNPs and mutations detected by Haloplex SureDesign Panel. ",
"section_name": "Additional file 2. Supplemental Data (Additional files 6 and 7).",
"section_num": null
},
{
"section_content": "The institutional review board of the University of California, San Diego (171884CX), and of the Fondazione Policlinico Agostino Gemelli (P/948/CE/ 2011) approved the research protocol.Samples were provided upon written informed consent. ",
"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
}
] |
10.1038/ncomms13765
|
Evolution of multiple cell clones over a 29-year period of a CLL patient
|
<jats:title>Abstract</jats:title><jats:p>Chronic lymphocytic leukaemia (CLL) is a frequent B-cell malignancy, characterized by recurrent somatic chromosome alterations and a low level of point mutations. Here we present single-nucleotide polymorphism microarray analyses of a single CLL patient over 29 years of observation and treatment, and transcriptome and whole-genome sequencing at selected time points. We identify chromosome alterations 13q14−, 6q− and 12q+ in early cell clones, elimination of clonal populations following therapy, and subsequent appearance of a clone containing trisomy 12 and chromosome 10 copy-neutral loss of heterogeneity that marks a major population dominant at death. Serial single-cell RNA sequencing reveals an expression pattern with high <jats:italic>FOS</jats:italic>, <jats:italic>JUN</jats:italic> and <jats:italic>KLF4</jats:italic> at disease acceleration, which resolves following therapy, but reoccurs following relapse and death. Transcriptome evolution indicates complex changes in expression occur over time. In conclusion, CLL can evolve gradually during indolent phases, and undergo rapid changes following therapy.</jats:p>
|
[
{
"section_content": "hronic lymphocytic leukaemia (CLL) is the most common B-cell malignancy in the US, Canada and Western Europe, and remains an incurable disease [1][2][3][4][5] .Recurrent somatic alterations include deletions of chromosomes 11q, 13q14 and 17p, and trisomy 12 (refs 6,7) and point mutations in SF3B1, NOTCH1 and TP53 (refs 8 -10).CLL represents an interesting model to study cancer progression, therapy response and relapse, as the disease is often detected many years before the initiation of treatment, and patients survive for a considerable time.We took advantage of an extremely rare situation of having yearly viably frozen tumour cells from a patient over the past 18 years of her 29-year disease course.By performing singlenucleotide polymorphism (SNP) array analysis at 16 yearly time points, as well as single-cell whole-genome sequence (WGS) and transcriptome, we have a detailed picture of molecular changes over time.During this time period, the patient had a 9-year period of indolent disease, a marked rise in white blood cell (WBC) counts, and multiple years of cytotoxic therapy with a moderate disease progression, followed by more rapid progression and chronic infections, and death.The resulting analysis provides an unparalleled look at cancer evolution over nearly 20 years. ",
"section_name": "C",
"section_num": null
},
{
"section_content": "Patient description.A female CLL patient was diagnosed in 1972 at age 47, with no evidence for cytogenetic abnormalities.We divide her disease into an early phase of observation (no treatment of disease lasting until 17 years after diagnosis), a middle phase (moderate disease progression requiring treatment, 18-25 years) and a late phase (disease progression, chronic infections and death, 26-29 years; Fig. 1b; Supplementary Fig. 1).Cytotoxic therapy (chlorambucil, an alkylating agent) administered in year 16, 22/23 resulted in a short-lived remission; eventually the patient progressed and died of her disease 29 years after diagnosis at age 76. SNP microarray analysis.To assess the sequence of changes in chromosomal abnormalities, we performed microarray analysis on tumour cells at 16 time points over 21 years (Figs 1a and2; Supplementary Figs 23456).There were no detectable aberrations at year 8 or 27 that reflect early disease and remission stages, respectively.Chromosome 6q and 13q deletions, copy-neutral loss of heterogeneity (LOH) on 10p and gain on chromosome 12 (years 10-12, 14, 17, 19-26 and 28) with at least two different events at each time point detected (Fig. 2; Supplementary Fig. 7). Chromosome 13q À was found for all 14 time points with alterations.The focal deletion region 13q14.3was identified at early time points and persisted to the end (years 10-28) and coincided with 6q À and 12q þ alterations.The large 13q deletion involving the RB1 gene (13q14.2) was found at later time points (years 20-25; Fig. 2; Supplementary Fig. 7), and may reflect disease progression and clonal selection.Chromosome 12 trisomy was found at later time points (years 25-28) and coincides with chromosome 10p copy number LOH (CNLOH; Fig. 2). Whole-genome and single-cell sequencing.To better understand the genomic changes, we sequenced the whole genome of unsorted peripheral blood mononuclear cells (PBMCs) DNA samples from years 10, 14, 21, 23, 24, 26 and 28 (Supplementary Table 1).The copy number variation (CNV) patterns were highly consistent with the results of the SNP microarray (Supplementary Figs 8,9).Interestingly, 6q deletion is always present with the 12q duplication (Fig. 3a).Copy-neutral LOH of chromosome 10p was found for four later time points (years 25-28) accompanied by whole chromosome 12 duplication (Fig. 3a).The disappearance of 6q deletion and 12q duplication, and the appearance of 10p CNLOH and 12 trisomy may reflect clonal selection in response to treatment.A low number of apparent somatic mutations were detected.These include S219C in MYD88 in 11-30% of reads in years 21, 23, 24 and 26, but undetectable in years 10, 14 and 28; and G49S in MED12 in 11% of reads only in year 26 (Supplementary Table 2).Both of these mutations have been previously detected in CLL 10 . To characterize the chromosome abnormalities at the singlecell level, low-coverage WGS from years 23 and 28 cells was generated (Fig. 1a; Supplementary Fig. 2; Supplementary Table 3) 11,12 .The data reveals clusters of tumour cells with chromosome 13q À , chromosome 6q À and partial or complex trisomy of chromosome 12 (Fig. 3b; Supplementary Fig. 10).The CNVs of 6q À , 12 þ , trisomy 12, focal 13q À and large 13q À detected by SNP microarray were all present in the single cells.However, single-cell analysis allows the CNVs to be resolved into five distinct sub-populations: normal-like; focal 13q À only; focal 13q À and large 13q À ; 6qÀ and 12q þ , 13q14.3À ; and trisomy 12 only (Fig. 3b).The chromosome 12q gain at year 23 always co-occurred with the 6q deletion consistent with the results of SNP microarray.These two CNVs were also accompanied by the focal 13q deletion.The remaining cells harboured either the large and focal 13q deletion or only the focal 13q deletion, for a total of three distinct cell populations.In year 28, the chromosome 12 trisomy emerged in the absence of other CNVs, indicating a novel origination of this clone.By combining the CNVs profiles from SNP array and single-cell sequencing, we reconstructed that the focal 13q14.3deletion appears first, followed by the co-occurring 6q À and 12q þ events (Fig. 3a).A new clone with the large 13q14.2-14.3deletion is first detected in year 20.Subsequently, the patient had multiple infections and elevated WBC counts, and after cytotoxic therapy (year 16, 22/23) and splenectomy in year 25, all chromosomal abnormalities were undetectable except the focal 13q13.3deletion.In year 24, both CNLOH of chromosome 10 and trisomy 12 emerge, both of which persist until year 28. Single-cell transcriptome analysis.To investigate gene expression profiles over time, we performed single-cell RNA transcriptome sequencing (RNA-seq) 13 on 300 unsorted tumour cells from the year of year 10, 20, 23, 26 and 28 (Supplementary Fig. 13; Supplementary Table 4).Unsupervised hierarchical clustering and principal component analysis (PCA) yielded six clusters (Fig. 4a; Supplementary Fig. 14).Cells from the earliest time point (year 10) are almost exclusively found in cluster D suggesting that this is the earliest detectable profile, and several cancer-related genes, including MAPK4 (ref.14), ERBB4 (ref.15) and PDGFRA 16 are in this cluster (Fig. 4b; Supplementary Tables 5 and6).In year 20, the cells adopt almost exclusively cluster F that contains several transcription factors involved in stem cell regulation, such as JUN, FOS, KLF4, KLF6 and CDKN1A, the MYD88 cascade (FOS, JUN, NFKBIA and RPS6KA5) or downstream signalling of the B-cell receptor (BCR; REL, CDKN1A and NFKBIA) 17,18 (Fig. 4b; Supplementary Tables 5 and6). Beginning in year 23, the cells developed a greater diversity of expression profiles divided into two major branches with clusters A, B and C, predominant in post-treatment samples.The cells from year 23 contain mostly profiles B and C; and in year 26, when the patient was in remission the cells largely reverted to cluster D. Finally, in year 28, at relapse, the cells again developed a great diversity with 56% of the cells adopting expression patterns clusters A and C, and 36% of cells a new expression profile, cluster E (Table 1; Fig. 4b; Supplementary Table 5). Therefore, the single-cell expression analysis reveals a diverse evolution of expression profiles over time and during treatment, remission and disease relapse. To further explore evolutionary models based on the single-cell RNA-seq data, we performed cell fate decision analyses using the Monocle program 19 to reorder the cells by their differential expression genes profiles.This pseudo-temporal ordering analysis decomposed the 300 cell profiles into three trajectories (Fig. 4c,d).The first trajectory is dominated by the oldest cells from year 10.The second trajectory contains mostly cells from year 20 and the third trajectory cells from year 23 followed by year 28 cells.The cells at the end of the third trajectory are mostly the year 28 cells from cluster E. The program Monocle groups genes with similar expression patterns along the pseudo-temporal trajectories.Several of these groups contain a high-low-high expression pattern, with high expression in early stages of leukaemia development, low expression after therapy and remission, and high upon relapse (Supplementary Fig. 15; Supplementary Table 7).These clusters are enriched in cancer-related pathways, such as BCR signalling, epidermal growth factor receptor (EGFR) signalling and extracellular signal-regulated kinase (ERK) signalling.Another group (group 7) is highly significantly enriched in cell cycle-related cyclin A and E genes, and NF-KappaB genes involved in B-cell development.This group displays low expression during the initial stages of leukaemogenesis and the highest expression at the early portion of trajectory 3. ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "In summary, we have performed multiple microarray, bulk and single-cell DNA and RNA analyses across the evolutionary lifespan of a single CLL patients' disease, response to therapy, relapse and death.We can divide the disease process into three phases: In the early phase, at the first time point, year 10 after clinical presentation of CLL, a focal deletion of chromosome 13q14.3was present along with a deletion of a portion of chromosome 6 and a gain of chromosome 12q.Single-cell analysis showed that the 6q À and 12q þ alterations are present in the same cells, which lack the chromosome 13 deletion (Fig. 5).This is consistent with data that 13q À cells can occur many years before CLL diagnosis (manuscript in preparation).The mosaic fraction of the 6q À and 12q þ alterations change in parallel over the period from year 10-19, consistent with the cell population containing these variants varying over time. In the middle phase, in year 20, the chr13 deletion expanded to include the RB1 gene, an event associated with poorer prognosis 20 , and a mutation in MYD88 (S219C) is also detected.These events are accompanied by a marked alteration in the gene expression pattern to expression cluster F. Elevated expression of FOS and JUN transcription factors and oncogenes, KLF4 (refs 21,22), one of the critical stem cell induction genes, as well as REL 23 , CDKN1A 24 and B-cell oncogenes are observed.This corresponds to the peak of a marked rise in WBC counts with the expansion of the chr13 deletion to include RB1 may lead to the altered regulation of one or more transcription factors, such as FOS/JUN and KLF4 accelerating the growth of the leukaemic cells. In the late phase, in year 22, further cytotoxic therapy was administered and in year 23 the expression pattern changed to one of high diversity with multiple different expression clusters present in different cell populations.Furthermore, in year 24 we see the appearance for the first time of a CNLOH event on chr10 and a complete trisomy of chr12, consistent with these events being concurrent in the same population.The single-cell sequencing shows that the trisomy 12 event occurred on an otherwise normal chromosome background. The patient required a splenectomy year 25, and in year 26 the gene expression pattern reverts to cluster D, the expression pattern present in the indolent phase.In year 27, most of the chromosome abnormalities become undetectable, and the patients' disease was stable.However, this state is short lived, and in year 28 all the chromosome abnormalities present before remission are detectable again.The patient suffered from increasing and recurrent respiratory tract infections and died in year 29.The gene expression pattern in year 28 shows a similar diversity in gene expression clusters as in 23, but the proportion of cells in each cluster has changed markedly.Therefore, we can follow the progression of the patients' disease at the molecular level along with the clinical changes or remission and relapse. To further understand the alterations in gene expression over time we used a new method, Monocle 19 , to analyse temporal changes in gene expression.Because cells do not synchronously move through expression states, this analysis can be used to reorder the cells based on both the time and expression state identifying genes that are coordinately expressed.This analysis showed cells from trajectory 1 contain many of the cells of year 10 and early time points.One group of cells branches off and stops (trajectory 2), and we interpret this as being cells eliminated by the therapy.Nearly, all the year 26 (cluster D) cells are in here.The third trajectory is composed mostly of cells with complex expression profiles from years 23 and 28.The final group of cells is almost exclusively from the year 28 time point and expression cluster E. Cluster E is a very minor component (2-7%) of the cells in year 10-26, but is 36% of the cells in the year 28.This suggests that these cells were rapidly proliferating at the end stage of disease.The cell population that contains the trisomy 12 and most likely the CNLOH event on chromosome 10, are likely the cluster E-expressing cells, containing FGF5, LYN, SETD2 and IL17RD.LYN 25 expressed in B cells and mediates response from the BCR.LYN is located on chromosome 12, and the trisomy 12 event may contribute to this overexpression.Deletions of chromosome 13 are the single most common somatic events in CLL.The focal 13q14.3deletion contains two long-non-coding RNA genes DLEU1 and DLEU2 and is thought to disrupt the expression of miR-16-1 and miR-15a (ref.26).This deletion is a good prognosis factor for CLL patients.However, large deletions that also contain RB1 are poor prognostic factors, especially when homozygous.While it has been assumed that the focal deletions expand to become the larger deletions, our data (Fig. 3c) provide direct evidence for this, as we can see individual single cells with transition states from the focal to expanded deletion.Interestingly, homozygous large deletion cells are almost completely absent from the single cells in year 28, indicating that they were eliminated by the therapy. The study is limited by involving a single patient, and therefore the conclusions to be drawn to other CLL subjects would require other analyses.However, this patient does have most of the commonly occurring chromosomal aberration found in CLL and therefore is a typical case in that respect.Our array and single-cell analyses over so many time points allows the reconstruction of the distribution and evolution of these chromosome abnormalities during the disease process.This patient had an indolent course of disease for many years, but once the disease required therapy, clinical progression proceeded in a typical manner.The most unique findings are the dramatic changes in gene expression patterns seen in single cells over time.Again it remains to be seen if this is typical of CLL from future single-cell multiyear analyses. In conclusion, we have performed, to our knowledge, the most detailed and extensive analysis of a single leukaemia patient in terms of number of samples analysed over time, using multiple DNA and RNA sequencing methods on bulk and single cells.We provide molecular analyses of the indolent disease state, acute disease, response to therapy and remission, and finally relapse.The percentage of single cells in each expression cluster as determined in Fig. 4a is shown at each time point tested.Bolded values are the highest at that date.Because the progression of CLL is in generally slow compared with other leukaemias and solid tumours, these disease stages can be dissected and provide insights into tumour progression and evolution.While our study has the limitation of following a single patient, we believe the insights are valuable to understanding cancer. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Patient information.The subject was diagnosed with CLL after a 1972 medical visit, when she presented with persistent upper respiratory infections (WBC ¼ 9,700; 47% lymphocytes).She received antibiotics and improved, but small posterior cervical and axillary nodes, and absent splenomegaly were noted on physical exam.Eventually, she received a referral to a haematologist who performed a sternal aspiration that revealed increased lymphocytes and a picture compatible with CLL.No therapy was required.At the time of her diagnosis, two brothers and her father also had a family history of CLL.Her WBC in 1973 ranged between 15-18,000 with 80% lymphocytes, and Hgb value of 13 g dl À 1 and platelets 250-300,000 cells per ml.Physical exam at that time showed no or minimal lymphadenopathy and no splenomegaly.Two years after diagnosis a bone marrow biopsy revealed mature lymphocytes with 'focal lymphoid aggregates' consistent with at diagnosis of CLL or well-differentiated lymphocytic lymphoma.Between 2 and 6 years after diagnosis she was followed with the only physical finding noted being a very small (o1 cm) right axillary lymph node.By year 8, her WBC had risen to 35,000 cells per ml, but she remained asymptomatic without additional physical findings.By year 13, mild splenomegaly was noted, with small lymph nodes persisting (anterior and posterior cervical, and right axilla).In year 16, with WBC ¼ 53,200 cells per ml, Hgb 11.2 g dl À 1 and platelets 114,000 cells per ml she received a short course of chlorambucil and prednisone.Her white count went from 56,000 to 25,000 cells per ml in year 17; platelets and Hgb remained stable B100,000 cells per ml and 11.0 g dl À 1 , respectively.Her spleen continued to slowly increase in size during this period and by late year 17 her counts had returned to earlier levels, WBC ¼ 53.2 cells per ml, Hgb ¼ 12.5 g dl À 1 , platelets ¼ 122,000 cells per ml and the spleen was measured 16 cm below the left costal margin.Otherwise, she continued to feel well with normal everyday activities.In year 19 following diagnosis she was involved in a serious car accident and suffered a splenic haematoma and other fractures (ankle and sternum).An ultrasound exam at that time revealed a 19 cm spleen.Her acute injuries and the haematoma resolved.Between year 22 and 23, she underwent another further treatment with chlorambucil and prednisone.This did not markedly change either her spleen size or WBC.By year 24, her WBC had risen to 80,000 cells per ml, Hgb 9 g dl À 1 and platelets 70,000 cells per ml.It was felt that her very large spleen was increasingly compromising her clinically, so on year 25 she underwent splenic embolization that was unsuccessful, followed by a splenectomy.The pathologic diagnosis of the spleen was 'splenomegalic CLL' and it measured (38.5 Â 24.7 Â 12.3 cm).She recovered and her WBC stabilized.Increasingly, she suffered from sinus and pulmonary infections, treated with rotating courses of antibiotics (levoquin, augmentin and cephlin) and monthly intravenous IG. In year 27, a breast biopsy was performed for a right breast mass and it revealed 'lymphocytes' consistent with CLL.WBC ¼ 38,300 cells per ml, Hgb ¼ 13 g dl À 1 and platelets ¼ 221,000 cells per ml.In year 28, increasing pulmonary infections and bronchiectasis were noted.Lymph nodes were unchanged.WBC ¼ 43,300 cells per ml, Hgb ¼ 13.3 g dl À 1 and platelets ¼ 210,000 cells per ml.The subject died in year 29, following diagnosis of increasing infectious complication of CLL.The studies were approved by the institutional review board of NIH, and informed consent was obtained from this subject. Cell staining for four-colour flow cytometry.The PBMCs from the CLL patient were stained using conjugated four-colour anti-human antibodies (fluorescein isothiocyanate, FITC-CD5; phycoerythrin, PE-CD3; allophycocyanin, APC-CD19 and peridininchlorophyll-protein cyanine 5.5, PerCP-CD45) in a single tube.Briefly, the PBMC cells were thawed in a 37 °C water bath, the cells washed twice using cold Flow Cytometry Staining Buffer Solution (eBioscience), centrifuged at 3.3g for 1 min, re-suspended in 100 ml of staining buffer solution and transferred to 5 ml tubes (Falcon).After addition of 5 ml of each antibody to 100 ml of cells, the cells were incubated at 4 °C in the dark for 40 min, centrifuged and re-suspended in 0.5 ml of staining buffer solution and kept on ice until sorting.Controls included (1) cells only; (2) CD3 (PE); (3) CD5 (FITC); (4) CD19 (APC); and (5) CD45 (PerCP-Cy5.5).All antibodies were obtained from eBioscience. The different target populations included (1) tumour B cells with CD19 þ /CD5 þ (population 7, P7); (2) normal T cells with CD3 þ /CD5 þ (population 5, P5); and (3) normal B cells with CD19 þ /CD5 À (population 8, P8; Supplementary Fig. 2).Cells were sorted on a Becton Dickenson FACSAria II with FACSDiva v6.1 software.The cell sorter was run with a 3,050 mW 488 nm solid state laser and a 40 mW 640 nm solid state laser.The detectors for FSC, SSC had 488/10 band-pass filters.These optical filters were used in front of the fluorescent detectors: 530/30 bp (FITC), 575/25 bp (PE), 710/50 bp (PerCP-Cy5.5) and 670/30 (APC).Compensations were calculated through the auto-compensation matrix using single-stained samples.The cells were sorted using a 70-mm nozzle run at a sample flow rate of 6,000-10,000 cells per second on a sort precision of 8-32-0.Both sample and collection tubes were maintained at 4 °C during the sort.Single cells were sorted into 96-well plates with the Automated Cell Deposition Unit run at a sample flow rate of 200-400 cells per second at a sort precision of 'single cell' (0-32-16).Only the sample was maintained at 4 °C during the 96-well plate sorts.Bulk-sorted cells were viably frozen in freeze medium in a programmable cell freezer and stored in liquid nitrogen until single-cell selection was performed.In most cases, we were able to collect more than one million tumour B cells, but normal T and normal B were obtained in only small quantities. Sorted cell DNA extraction.Extraction of DNA from sub-populations of cells was performed using the ZR-Duet DNA/RNA MiniPrep kit (ZYMO Research).The sample which contained normal T cells from year 10 and 28 was removed from analysis due to low quality. Single-cell RNA sequencing.All complementary DNA products of single CLL B cells in this study were prepared by a microwell based a platform called MIR-ALCS 13 .A total of 1 ng complementary DNA product from each cell was used for library construction using the TruePrepTM Mini DNA Sample Prep Kit (Vazyme Biotech) followed by barcode labelling.A total of 362 single cells were sequenced on Illumina Hiseq 2000 sequencing system with SE50 sequencing strategy (Supplementary Table 4).Of these cells, 38 cells were from year 10, 48 cells from year 20, 126 cells from year 23, 35 cells from year 26 and 115 cells from year 28. Single-cell DNA sequencing.Single CLL tumour cells are selected by a Micromanipulator (Eppendorf TransferMan NK2) under the inverted fluorescence microscope (Olympus IX-71), and pipetted into PCR tubes that contained 2 ml lysis buffer.Single-cell DNA amplification was carried out using the REPLI-g Single Cell Kit (Qiagen), and amplified DNA with positive results for at least six out of eight house-keeping genes were selected for subsequent library construction.DNA was fragmented with the Covaris E-210 ultrasonicator with adjusted shearing parameters.We purified the DNA fragments, blunted the ends, added A tails and ligated them with adaptors to prepare Hiseq libraries.A total of 116 single cells were sequenced on Illumina Hiseq 2000 sequencing system with PE 100 sequencing strategy (Supplementary Table 3).Of these cells, 54 cells were from year 23 and 62 cells were from year 28. Bulk DNA sequencing.DNA extracted from sorted cell population of year 10, 14, 21, 23, 24, 26 and 28 by using DNeasy Blood & Tissue Kit (Qiagen).We used Covaris LE220 (Covaris) to break the genome DNA into fragments with a 350 bp peak by optimal shearing parameters.We purified the fragments by using Agencourt AMPure XP beads (Beckman Coulter).We repaired the ends of fragments, added A tails and ligated the adaptors.After fragment size selection by gel, we did 10 cycles of PCR to enrich the fragments.After purification by Agencourt AMPure XP beads (Beckman Coulter), we did the quality control of libraries by Agilent 2100 Bioanalyzer (Agilent Technologies) and quantitative PCR using ABI StepOne Plus Real-Time PCR system (Life Technologies).Paired-end 150 bp length reads were sequenced on Illumina Hiseq 4000 sequencing system.SNP array methods.Genomic DNA was extracted from unsorted bulk cell DNA, as well as sorted tumour B cells, and normal T and B cells.Genomic DNA was screened and analysed at the NCI according to the standard sample handling process of the Cancer Genomics Research Laboratory, Division of Cancer Epidemiology and Genetics before genotyped using Illumina Infinium OmniExpress BeadArray assays.Sample intensity files (two files per sample, for red and green channels) were loaded into the Illumina GenomeStudio software.The intensity data were normalized using the Illumina five-step self-normalization procedure, which used information contained in the array itself to convert raw X and Y (allele A and allele B) signal intensities to normalized values.The chromosome mosaicism was detected using log R ratio (LRR) and B allele frequency (BAF).The LRR value is the normalized measure of total signal intensity and provides data on relative copy number.The BAF derived from the ratio of allelic probe intensity is the proportion of hybridized sample that carries the B allele as designated by the Illumina Infinium Assay.The LRR and BAF values for each assay were exported from GenomeStudio software using the 'Genotype Final Report' format. Quantile normalization was applied to remove dye bias and improves the asymmetry in the detection of the two alleles for each SNP, which influences both allelic proportions and copy number estimates.GC/CPG correction was applied to reduce the wavy patterns of signal intensities and improves the accuracy of CNV detection.The LRR and BAF were re-estimated on the quantilenormalized and GC/CpG corrected values.All of these procedures were implemented using GLU software package (http://code.google.com/p/glu-genetics/) that was developed at Cancer Genomics Research Laboratory.The renormalized LRR and BAF values from qualifying assay were then analysed using custom software pipelines that involved BAF segmentation packages (http://baseplugins.thep.lu.se/wiki/se.lu.onk.BAFsegmentation) to detect mosaic copy number aberration with minimum of 20 probes per segment to minimize were the final results used in the paper (Fig. 4a).The cells were then divided into six groups (A-F cluster) according to the hierarchical clustering, and we used the hypergeometric distribution test to identify the marker genes.The P values were adjusted using the 'BH' method, and genes with q valueso0.01were considered as particularly highly expressed marker genes in the A-F clusters (Supplementary Table 5).We did not detect significantly expressed genes in cluster C. Pseudo-temporal analysis.To further investigate the gene expression changes during tumour evolution and the relationships between different cells from different time points, we performed pseudo-temporal analysis using the R package Monocle 19 .We first detected the differentially expressed genes according to corresponding time point of each cell using Monocle and 3,546 genes with a Po0.01 were retained for further analysis.The pseudo-temporal path number was set at 2, and the cells were divided into three trajectories.Considering that the cells were not in a strict developmental state and that the intervals between different time points were very long, we modified the pseudo-temporal ordering of each cell.Cells were divided in to three groups according to the inferred pseudotemporal phases, and the relative orderings of cells in the same group remained unchanged.The group containing the most cells from the earliest time (year 10) was set to be the start of the pseudo-temporal path, and the group containing the most cells from year 23 and 28 was set as the end of the path (Fig. 4c,d).The last group was placed between the two groups.We then clustered the genes into 10 groups to make sure that the gene expression along the inferred pseudotemporal path reflected the changes along the actual time (Supplementary Fig. 15).Therefore, we could combine the pseudo-temporal ordering and the actual time to investigate the gene expression changes and their potential influences.Reactome enrichment was performed on the genes from each group using the R package ReactomePA 48 with the 9,258 genes as the background and a q value cutoff of 0.05 (Supplementary Table 7).We did not detect enriched pathways in cluster 6. ",
"section_name": "Methods",
"section_num": null
}
] |
[
{
"section_content": "We thank Rongchang Chen, Weijian Rao, Xiaolong Zhang, Jie Wang, Zhanlong Mei, Xinlan Zhou, Nannan Li, Xulian Shi and Fatima Abbassi for help on data analysis and discussion, Kathleen Noer and the CCR Flow Cytometry Core for cell sorting.This project was supported by grants of the Shenzhen Science and Technology Program (CXZZ20150330171838997), the Shenzhen Municipal Government of China (ZDSYS20140509153457495) and the Science, Technology and Innovation Committee of Shenzhen Municipality (JSGG20140702161347218).This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E.The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Data availability.Raw data have been uploaded to the database of Genotypes and Phenotypes (dbGAP, http://www.ncbi.nlm.nih.gov/gap/)under the study accession code phs001177, SRA399097.This study includes all sequencing data from WGS and whole-transcriptome sequencing.The remaining data supporting the finding of this study are contained within the article and the Supplementary Information files, or available from the authors on request. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "the false discovery.The copy number status was assigned (mosaic loss, mosaic gain and mosaic copy-neutral LOH) and mosaic proportion of abnormal cells was estimated.All potential events were plotted.False positive calls were excluded from analysis base on manual review on each plot. To prevent the lack of detection of the chromosome 13q14.3focal deletion, due to the small size of this region, a t-test (two sided) was used to target the region between bases 49,139,793 and 50,269,706 (GRCh36).The LRR and BAF were examined for deviations from expected LRR and BAF.For each time point, an unequal variance t-test was applied to find mean LRR value and mean BAF for the tested region that is significantly different from mean LRR values and mean BAF on chromosome 1 (used as the reference).Results from t-test were plotted and manually reviewed to confirm a 13q14.3 Public data set access.The human (Homo sapiens) reference genome sequence (Hg19, GRCh37) was downloaded from the University of California Santa Cruz Genome Bioinformatics (http://genome.ucsc.edu/).The transcriptome reference annotation GTF file 27 was downloaded from http://www.ensembl.org/.The GTF file retained the information of autosomes and sex chromosome X before further analysis.The known SNPs and indels in 1000 Genomes Project and dbSNP v137 (ref.28) were downloaded from ftp://ftp.broadinstitute.org/. Genome alignment variant calling.Reads resulting from Illumina Hiseq 4000 sequencing were aligned to hg19 (UCSC) using BWA (v0.7.12-r1044) 29 with parameters 'mem -t 8 À P -M' and the generated files were then sorted and PCR duplicates removed using Picard (v1.72) (http://broadinstitute.github.io/picard).Bamtools (v2.1.1) 30was used to filter out multi-mapped reads and those with mapping quality o1.Subsequently, the BAM files were indexed by samtools (v0.1.18) 31. SNPs and indels calling.We detected SNPs and indels according to the GATK 32 best practice workflows.After duplicate-read removal using Picard, the BAM files were recalibrated and variants were called using GATK v3.3.0 with known indels in the 1000 Genomes Project 33 and known SNPs in dbSNP v137 (ref.28).Then we built the index of each VCF file, compressed them using tabix (v0.2.6) 34 and merged all SNPs in the VCF files using VCFtools (v0.1.12b) 35.The SNPs were filtered using GATK with parameters '-T VariantFiltration --filterExpression 'QDo2.0|| FS460.0 || MQo40.0'--filterName 'snp_filter' --clusterSize 2 --clusterWindowSize 10'. Minor allele frequency analysis.Because there is no normal sample to use as a control, we could not detect the precise SNVs of the tumour samples.Therefore, we tried to eliminate possible germline SNPs and analysed the minor allele frequency changes from year 10-28.We annotated all SNPs using ANNOVAR 36 with parameters '-protocol refGene, popfreq_all_20150413, snp138NonFlaggedoperation g,f,f'.The SNPs with allele frequency40.0001 in 1000G_AMR and ExAC_AMR data sets and the SNPs marked as snp138NonFlagged were removed from further analysis.To obtain a more confident SNP allele frequency, we retained only those SNPs with depth Z30 in all seven samples.A total of 6,149 SNPs were retained.We then performed the PCA analysis using the R package FactoMineR 37 , and filtered out the sites with PC14 À 1 (Supplementary Fig. 16a,b), which showed no significant difference among each other and were thought to be possible germline SNPs. The remained 1,065 SNPs were clustered using SOTA in R package clValid 38 and divided into 12 groups.Of the 12 clusters, 3 clusters were removed because the minor allele frequency (MAF) changed in a small range among all samples and were considered as possible germline SNPs (Supplementary Fig. 16c).Most of these SNVs are in introns and non-coding regions and are not likely to affect cancer progression.By clustering the SNVs with similar MAF pattern, we found SNVs (cluster 4) that are lost in later time points and may represent regions of deletion (Supplementary Fig. 17).Other SNVs increased in frequency and then disappeared perhaps represent variants in cells eliminated during treatment.Some SNVs showed increasing MAF during the later stages and may represent accumulated passenger mutations. SV detection in bulk samples.After duplicate-read removal using Picard, structural variations were detected for each sample separately using CREST 39 with Hg19 as the reference genome.Then, the generated tumour.predSV.txtfiles were annotated using ANNOVAR with Hg19 and dbSNP v137.For an alternative method to call CNVs, we removed deletions and amplifications from the results of CREST.SVs with supporting reads o3 in each sample were filtered out.But we did not detect any high confidence rearrangements of known cancer genes (Supplementary Table 8). Genome alignment of bulk and single-cell samples for CNV calling.Reads resulting from Hiseq 2000 sequencing were aligned to Hg19 (UCSC) using Bowtie (v1.0.0) 40 with parameters '-S -t -m 1 --best -strata'.Then we transformed the subsequent SAM files to BAM format and sorted them using samtools (v0.1.19).Afterwards, we also used samtools (v0.1.19) to remove PCR duplicates and build an index for each BAM file. CNVs calling in bulk and single-cell samples.Copy number was computed for each sample separately using a modified method based on that developed by the Cold Spring Harbour Laboratory 41,42 .We followed their bioinformatic workflow to detect CNVs and used the Python script provided to generate the 'bin boundaries' file for 10,000 bins in Hg19 suitable for 150-bp length reads.For single-cell samples, the 'bin boundaries' file had 10,000 bins and was suitable for 100-bp length reads.We calculated the read number and the ratios based on the average read number in each bin of all bulk and single-cell samples.After GC correction, the R package DNAcopy 43 was used to calculate the copy number ratios and merge bins into segments.Bins in the same segment had the same segment ratio and segment ratios equal to 1 represent normal.The ratios 41 represent amplification, and the rations o À 1 represent deletion.The GC corrected ratios of each bin and the inferred rations of segments of specific chromosomes were extracted to display the detailed copy number changes for bulk and single-cell samples (Supplementary Figs 9,11,12). Single-cell WGS filtering and clustering.The Median Absolute Pairwise Difference (MAPD) 44 measures the absolute difference between the log2 copy number ratios of every pair of neighbouring bins and then takes the median across all bins.Higher MAPD scores reflect greater noise, typically associated with poor-quality samples.After obtaining the copy number of each bin for all singlecell-genome sequencing samples, we adopted the MAPD algorithm to filter out 14 samples with MAPD Z1.4.Then, we transformed the segment ratios of the remained cells into a log2-scale, and set the log2-scaled ratios with values41 or o À 1 to 1 or À 1, respectively.The transformed segment ratios were used to the hierarchical clustering analysis using R package NMF 45 with the clustering method of 'ward.D2'. False positives in single-cell CNV profiles.When we clustered the single cells using the CNVs, we found two cells with 6q deletions that had a larger amplification in chr12 than other cells (Fig. 3b).We extracted the raw ratios of each bin before the segment ratio calling, and found the ratios showed significant difference between the different portions in the chr12 amplification region in the same cells (Student's t-test, two sided, Supplementary Fig. 12).Although the differences of raw ratios between the true amplification region and the false positive region were significant, we speculated that the difference were not significant enough for the CNV detection methods.Therefore, the method merged the adjacent bins to the actual amplification region to generate a larger CNV, leading to a false positive result. We also found two abnormal cells when we clustering the cells with the three main CNV regions (chr6, chr12 and chr13).We extracted the CNV profiles of the whole chromosomes, and found that both of these two cells harboured larger CNVs.One cell harboured deletion of all chr6 and 13 and is trisomy 12 (Supplementary Fig. 11a-d) and the other cell, chr6 and 13 deletion and normal chr12 (Supplementary Fig. 11e-h).We removed these two cells from the hierarchical clustering analysis of the three specific CNVs shown in Fig. 3b. Single-cell RNA-seq data processing.Reads resulting from Hiseq 2000 sequencing were filtered using an in-house C þ þ script, and aligned using TotHat2 (ref.46).The fragments per kilobase of exon per million fragments mapped (FPKM) values were calculated using the R package edgeR 47 .The detailed process and parameters were described in a recent publication 13 .The single cells with less than one million reads mapping to gene regions were removed from further analysis. Filtering of genes and samples.Cells with expressed genes (FPKM41) 48,000 were also filtered out, as they showed much higher expression levels than the average and likely contain more than one cell.To reduce the potential influence of RNA degradation, we only retained the protein-coding genes.We then picked a set of genes that were expressed in at least half of all cells with FPKM41 and calculated the expressed gene number of every cell.We excluded from the analysis any cells that expressed below the 10th percentile of that gene set and whose mean FPKMr1.We also removed genes with the mean FPKM more than the 99th percentile for all genes.In total, 300 single cells and 9,258 genes remained for further analysis out of 362 total cells sequenced. PCA and hierarchical clustering analysis.We used an in-house R script to perform the PCA and hierarchical clustering.The PCA analysis was performed on all the cells and genes after filtering using the R package FactoMineR 37 .A total of 80 genes, the first 20 genes of the first four principle components, were used to perform the hierarchical clustering.The cells were divided into six groups according to the result of the hierarchical clustering.We then performed the Kruskal-Wallis rank sum test (KW test) and adjusted the P values using 'fdr' method.We retained the genes with q valueo0.01.The remaining genes were used to perform the PCA, hierarchical clustering analysis and KW test.Genes with q valueo0.01 were used for the third PCA and hierarchical clustering analysis, which 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: Zhao, Z. et al.Evolution of multiple cell clones over a 29-year period of a CLL patient.Nat.Commun.7, 13765 doi: 10.1038/ncomms13765 (2016). 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) 2016 ",
"section_name": "focal deletion.",
"section_num": null
},
{
"section_content": "the false discovery.The copy number status was assigned (mosaic loss, mosaic gain and mosaic copy-neutral LOH) and mosaic proportion of abnormal cells was estimated.All potential events were plotted.False positive calls were excluded from analysis base on manual review on each plot. To prevent the lack of detection of the chromosome 13q14.3focal deletion, due to the small size of this region, a t-test (two sided) was used to target the region between bases 49,139,793 and 50,269,706 (GRCh36).The LRR and BAF were examined for deviations from expected LRR and BAF.For each time point, an unequal variance t-test was applied to find mean LRR value and mean BAF for the tested region that is significantly different from mean LRR values and mean BAF on chromosome 1 (used as the reference).Results from t-test were plotted and manually reviewed to confirm a 13q14.3 ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Public data set access.The human (Homo sapiens) reference genome sequence (Hg19, GRCh37) was downloaded from the University of California Santa Cruz Genome Bioinformatics (http://genome.ucsc.edu/).The transcriptome reference annotation GTF file 27 was downloaded from http://www.ensembl.org/.The GTF file retained the information of autosomes and sex chromosome X before further analysis.The known SNPs and indels in 1000 Genomes Project and dbSNP v137 (ref.28) were downloaded from ftp://ftp.broadinstitute.org/. Genome alignment variant calling.Reads resulting from Illumina Hiseq 4000 sequencing were aligned to hg19 (UCSC) using BWA (v0.7.12-r1044) 29 with parameters 'mem -t 8 À P -M' and the generated files were then sorted and PCR duplicates removed using Picard (v1.72) (http://broadinstitute.github.io/picard).Bamtools (v2.1.1) 30was used to filter out multi-mapped reads and those with mapping quality o1.Subsequently, the BAM files were indexed by samtools (v0.1.18) 31. SNPs and indels calling.We detected SNPs and indels according to the GATK 32 best practice workflows.After duplicate-read removal using Picard, the BAM files were recalibrated and variants were called using GATK v3.3.0 with known indels in the 1000 Genomes Project 33 and known SNPs in dbSNP v137 (ref.28).Then we built the index of each VCF file, compressed them using tabix (v0.2.6) 34 and merged all SNPs in the VCF files using VCFtools (v0.1.12b) 35.The SNPs were filtered using GATK with parameters '-T VariantFiltration --filterExpression 'QDo2.0|| FS460.0 || MQo40.0'--filterName 'snp_filter' --clusterSize 2 --clusterWindowSize 10'. Minor allele frequency analysis.Because there is no normal sample to use as a control, we could not detect the precise SNVs of the tumour samples.Therefore, we tried to eliminate possible germline SNPs and analysed the minor allele frequency changes from year 10-28.We annotated all SNPs using ANNOVAR 36 with parameters '-protocol refGene, popfreq_all_20150413, snp138NonFlaggedoperation g,f,f'.The SNPs with allele frequency40.0001 in 1000G_AMR and ExAC_AMR data sets and the SNPs marked as snp138NonFlagged were removed from further analysis.To obtain a more confident SNP allele frequency, we retained only those SNPs with depth Z30 in all seven samples.A total of 6,149 SNPs were retained.We then performed the PCA analysis using the R package FactoMineR 37 , and filtered out the sites with PC14 À 1 (Supplementary Fig. 16a,b), which showed no significant difference among each other and were thought to be possible germline SNPs. The remained 1,065 SNPs were clustered using SOTA in R package clValid 38 and divided into 12 groups.Of the 12 clusters, 3 clusters were removed because the minor allele frequency (MAF) changed in a small range among all samples and were considered as possible germline SNPs (Supplementary Fig. 16c).Most of these SNVs are in introns and non-coding regions and are not likely to affect cancer progression.By clustering the SNVs with similar MAF pattern, we found SNVs (cluster 4) that are lost in later time points and may represent regions of deletion (Supplementary Fig. 17).Other SNVs increased in frequency and then disappeared perhaps represent variants in cells eliminated during treatment.Some SNVs showed increasing MAF during the later stages and may represent accumulated passenger mutations. SV detection in bulk samples.After duplicate-read removal using Picard, structural variations were detected for each sample separately using CREST 39 with Hg19 as the reference genome.Then, the generated tumour.predSV.txtfiles were annotated using ANNOVAR with Hg19 and dbSNP v137.For an alternative method to call CNVs, we removed deletions and amplifications from the results of CREST.SVs with supporting reads o3 in each sample were filtered out.But we did not detect any high confidence rearrangements of known cancer genes (Supplementary Table 8). Genome alignment of bulk and single-cell samples for CNV calling.Reads resulting from Hiseq 2000 sequencing were aligned to Hg19 (UCSC) using Bowtie (v1.0.0) 40 with parameters '-S -t -m 1 --best -strata'.Then we transformed the subsequent SAM files to BAM format and sorted them using samtools (v0.1.19).Afterwards, we also used samtools (v0.1.19) to remove PCR duplicates and build an index for each BAM file. CNVs calling in bulk and single-cell samples.Copy number was computed for each sample separately using a modified method based on that developed by the Cold Spring Harbour Laboratory 41,42 .We followed their bioinformatic workflow to detect CNVs and used the Python script provided to generate the 'bin boundaries' file for 10,000 bins in Hg19 suitable for 150-bp length reads.For single-cell samples, the 'bin boundaries' file had 10,000 bins and was suitable for 100-bp length reads.We calculated the read number and the ratios based on the average read number in each bin of all bulk and single-cell samples.After GC correction, the R package DNAcopy 43 was used to calculate the copy number ratios and merge bins into segments.Bins in the same segment had the same segment ratio and segment ratios equal to 1 represent normal.The ratios 41 represent amplification, and the rations o À 1 represent deletion.The GC corrected ratios of each bin and the inferred rations of segments of specific chromosomes were extracted to display the detailed copy number changes for bulk and single-cell samples (Supplementary Figs 9,11,12). Single-cell WGS filtering and clustering.The Median Absolute Pairwise Difference (MAPD) 44 measures the absolute difference between the log2 copy number ratios of every pair of neighbouring bins and then takes the median across all bins.Higher MAPD scores reflect greater noise, typically associated with poor-quality samples.After obtaining the copy number of each bin for all singlecell-genome sequencing samples, we adopted the MAPD algorithm to filter out 14 samples with MAPD Z1.4.Then, we transformed the segment ratios of the remained cells into a log2-scale, and set the log2-scaled ratios with values41 or o À 1 to 1 or À 1, respectively.The transformed segment ratios were used to the hierarchical clustering analysis using R package NMF 45 with the clustering method of 'ward.D2'. False positives in single-cell CNV profiles.When we clustered the single cells using the CNVs, we found two cells with 6q deletions that had a larger amplification in chr12 than other cells (Fig. 3b).We extracted the raw ratios of each bin before the segment ratio calling, and found the ratios showed significant difference between the different portions in the chr12 amplification region in the same cells (Student's t-test, two sided, Supplementary Fig. 12).Although the differences of raw ratios between the true amplification region and the false positive region were significant, we speculated that the difference were not significant enough for the CNV detection methods.Therefore, the method merged the adjacent bins to the actual amplification region to generate a larger CNV, leading to a false positive result. We also found two abnormal cells when we clustering the cells with the three main CNV regions (chr6, chr12 and chr13).We extracted the CNV profiles of the whole chromosomes, and found that both of these two cells harboured larger CNVs.One cell harboured deletion of all chr6 and 13 and is trisomy 12 (Supplementary Fig. 11a-d) and the other cell, chr6 and 13 deletion and normal chr12 (Supplementary Fig. 11e-h).We removed these two cells from the hierarchical clustering analysis of the three specific CNVs shown in Fig. 3b. Single-cell RNA-seq data processing.Reads resulting from Hiseq 2000 sequencing were filtered using an in-house C þ þ script, and aligned using TotHat2 (ref.46).The fragments per kilobase of exon per million fragments mapped (FPKM) values were calculated using the R package edgeR 47 .The detailed process and parameters were described in a recent publication 13 .The single cells with less than one million reads mapping to gene regions were removed from further analysis. Filtering of genes and samples.Cells with expressed genes (FPKM41) 48,000 were also filtered out, as they showed much higher expression levels than the average and likely contain more than one cell.To reduce the potential influence of RNA degradation, we only retained the protein-coding genes.We then picked a set of genes that were expressed in at least half of all cells with FPKM41 and calculated the expressed gene number of every cell.We excluded from the analysis any cells that expressed below the 10th percentile of that gene set and whose mean FPKMr1.We also removed genes with the mean FPKM more than the 99th percentile for all genes.In total, 300 single cells and 9,258 genes remained for further analysis out of 362 total cells sequenced. PCA and hierarchical clustering analysis.We used an in-house R script to perform the PCA and hierarchical clustering.The PCA analysis was performed on all the cells and genes after filtering using the R package FactoMineR 37 .A total of 80 genes, the first 20 genes of the first four principle components, were used to perform the hierarchical clustering.The cells were divided into six groups according to the result of the hierarchical clustering.We then performed the Kruskal-Wallis rank sum test (KW test) and adjusted the P values using 'fdr' method.We retained the genes with q valueo0.01.The remaining genes were used to perform the PCA, hierarchical clustering analysis and KW test.Genes with q valueo0.01 were used for the third PCA and hierarchical clustering analysis, which ",
"section_name": "focal deletion.",
"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. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/ How to cite this article: Zhao, Z. et al.Evolution of multiple cell clones over a 29-year period of a CLL patient.Nat.Commun.7, 13765 doi: 10.1038/ncomms13765 (2016). 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) 2016 ",
"section_name": "Additional information",
"section_num": null
}
] |
10.1186/s12967-022-03649-4
|
Loss of endothelial EMCN drives tumor lung metastasis through the premetastatic niche
|
<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>Metastasis is the primary cause of cancer-related mortality. Metastasis involves a complex multistep process during which individual tumor cells spread primarily through destruction of the endothelial barrier, entering the circulatory system to colonize distant organs. However, the role of the endothelial barrier as the rate-limiting process in tumor metastasis and how these processes affect the regulation of the host microenvironment at the molecular level are poorly understood.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>Here, we analyzed differentially expressed genes in breast cancer and lung adenocarcinoma, including metastatic and recurrent specimens, using TCGA dataset. The effects of EMCN on endothelial cells in vitro and in vivo were analyzed by assessing angiogenesis and vascular permeability, respectively. We established a syngeneic mouse model of endothelial cell-specific knockout of EMCN (EMCN<jats:sup>ecko</jats:sup>) to study the role of EMCN in tumor growth and metastasis. Transcriptome sequencing, Western blotting, qPCR and immunofluorescence confirmed important factors in the premetastatic niche. A mouse model of allograft tumor resection with lung metastasis was established to confirm the therapeutic effect of a notch inhibitor combined with an anti-TGF-β antibody.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>We found a strong correlation of EMCN deficiency with tumor recurrence and metastasis. Comparative experiments in WT and EMCN<jats:sup>ecko</jats:sup> mice revealed that endothelial EMCN deficiency did not affect primary tumor growth significantly but strongly promoted spontaneous metastasis. EMCN deficiency was associated with gene profiles that regulate cell junctions in vitro and enhance vascular permeability in vivo. Mechanistically, EMCN deficiency mainly affected the host microenvironment and led to the formation of a lung premetastatic niche by recruiting Ly6G<jats:sup>+</jats:sup> neutrophils and upregulating MMP9, S100A8/A9 and TGF-β expression. Anti-TGF-β antibody effectively eliminated TGF-β-induced neutrophil polarization, thereby reducing lung metastasis. Notably, the combination of a Notch inhibitor and an anti-TGF-β antibody effectively inhibited tumor growth and lung metastasis and prolonged the survival time of mice.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>We present a new translational strategy of EMCN as a new key player in tumor lung metastasis by affecting the host microenvironment. These findings could provide a sound theoretical basis for clinical treatment.</jats:p> </jats:sec>
|
[
{
"section_content": "Metastasis of primary malignant tumors is the main cause of cancer-related mortality [1].Only a few effective treatment options are available for patients with cancer metastasis [2].Metastasis is a gradual process that includes the invasion and dissemination of malignant cells, colonization by circulating tumor cells, formation of a premetastatic niche and adaptation to the microenvironment of the metastatic site.Growing evidence shows that tumor metastasis colonization depends not only on abnormal gene changes in cancer cells but also on the premetastatic niche.Disseminated cancer cells successfully colonize distant organs by altering the local microenvironment to survive [3].Therefore, interventions targeting the premetastatic niche may represent a new strategy for inhibiting tumor metastasis and therapeutic intervention for patients with metastatic cancers. Neutrophils are the most abundant leukocytes (50-70%) in human blood circulation.Early studies have shown that neutrophils play a key role in inflammation and host resistance to microbial infections [4].Growing evidence shows that neutrophils also play a significant role in tumor progression.However, neutrophils have been shown to possess both protumor and antitumor properties, but this finding is controversial.Lev Becker et al. found that the neutrophil-derived antitumor molecule ELANE can selectively kill tumor cells and attenuate tumorigenesis [5].It has also been found that the interaction between neutrophils and circulating tumor cells in blood promotes the cell cycle progression and the metastatic potential of circulating tumor cells [6].The inflammatory factors released by neutrophils stimulated by ovarian tumors become neutrophil extracellular traps and promote the formation of a premetastatic niche and ovarian cancer cell metastasis [7]. Endothelial cells form blood vessels, support tumor growth by providing nutrition and oxygen, and play an important role in cancer metastasis [8].These cells provide vascular secretory factors to coordinate tumor progression.Constitutive activation of vascular Notch signaling promotes metastasis by activating proinflammatory and senescence signaling in endothelial cells [9].EMCN is a transmembrane O-sialylated protein expressed on the surface of the endothelium.Human and mouse EMCN contain 261 amino acid residues and possess an extracellular domain rich in serine and threonine residues [10,11].Functionally, EMCN has been reported to affect tube morphogenesis of endothelial cells in vitro and leukocyte adhesion to endothelial cells in the blood [12,13].EMCN/MUC15 combined analysis has been suggested as a prognostic signature of gastric cancer [14].Endothelial Notch activation downregulates EMCN and promotes the cross endothelial migration of neutrophils in vitro, thus modulating acute inflammation in hepatic ischemia/reperfusion injury [15]. Herein, we report a new mechanism by which EMCN deficiency leads to tumor metastasis independent of tumor growth.EMCN deficiency is associated with gene profiles related to the regulation of cell junctions in vitro and vascular permeability in vivo.Furthermore, we demonstrate that EMCN deficiency leads to the formation of a premetastatic niche to promote tumor metastasis in a neutrophil-dependent manner.A Notch inhibitor combined with an anti-TGF-β antibody attenuated tumor metastasis.Of note, TCGA data showed that patients with high EMCN or low Notch1 expression survived longer than those with low EMCN or high Notch1 expression, indicating that EMCN and Notch levels have therapeutic and prognostic potential. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "EMCN flox/flox mice and Tek-CreERT2 mice were purchased from Beijing VIEWSOLID Biotechnology Co., Ltd., and raised in a temperature-controlled facility with a 12-h light/dark cycle in a specific pathogen-free environment in a separate ventilation cage.Mice had ad libitum access to food and water.All animal experiments were approved by the Animal Experiments Committee of the Chinese Academy of Medical Sciences (IACUC: GR21003).EMCN flox/flox mice were crossed with Tek-CreERT2 mice to generate endothelial cellspecific EMCN knockout mice (EMCN ecko ).All female or male mice aged 8-10 weeks (weight 23-25 g) were randomly divided into groups for follow-up experiments.All genotyping was confirmed by PCR.Gene deletion by Cre recombinase was achieved by intraperitoneal (i.p.) injection of tamoxifen (75 mg/kg body weight) (Sigma-Aldrich, CAS# 10540-29-1) every day for 5 days, starting seven days before tumor cell injection.EMCN knockout efficiency was determined by Western blot. ",
"section_name": "Animal models",
"section_num": null
},
{
"section_content": "LLC (LL/2) murine lung carcinoma cells, B16-F10 melanoma cells and PUMC-HUVEC-T1 (later HUVEC, SV40T transformed immortalized human umbilical vein endothelial cells) were purchased from the National Infrastructure of Cell Line Resource and maintained in DMEM (Gibco) supplemented with 10% FBS (Gibco).The human salivary gland adenoid cystic carcinoma cell line SACC-LM (highly invasive) was obtained from the Peking University Hospital of Stomatology.The cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and a 1% penicillin-streptomycin solution (Invitrogen).Cell line authentication was performed by short tandem repeat (STR) assessment.All experiments were performed with mycoplasma-free LLC-luciferase clones produced by continuous puromycin (800 μg/ml) screening of LLC cells overexpressing the lentiviral-driven luciferase gene.For EMCN deletion, three human shRNA sequences were cloned into a plasmid vector, and lentivirus was packaged by Shanghai Jikai GENE Biological Company.HUVECs were infected with lentivirus (MOI = 20).HUVEC cell lines with stable EMCN knockdown were screened by puromycin.Negative control and EMCN knockdown were termed HUVEC/shNC and HUVEC/shEMCN, respectively.HUVEC proliferation was analyzed by CCK8 kits (Dojindo, C0038).For angiogenesis, the concentrated and reduced growth factor matrix gel (Corning) was placed into a 24-well plate, and the plate was incubated at 37 °C for 30 min.HUVECs/shNC and HUVECs/shEMCN (1 × 10 5 cells) suspended in serum-containing media were added to 24-well plates with solidified Matrigel.Images were obtained with a contrast microscope and analyzed using the Fiji plug-in for ImageJ software. ",
"section_name": "Cell culture, proliferation and angiogenesis",
"section_num": null
},
{
"section_content": "HUVECs were infected with lentivirus (MOI = 20).Cells with stable NICD knockdown were selected in puromycin-supplemented medium.NICD knockout efficiency and EMCN expression were detected by Western blotting.Condition medium (CM) was prepared by seeding 1 × 10 6 LLC, SACC-LM and B16-F10 cells in common culture medium supplemented with 10% FBS for 48 h.The harvested CM was then centrifuged at 250g for 10 min to remove cells and debris.Then, HUVECs were washed with PBS and incubated further for 48 h in different tumor cell-conditioned media. ",
"section_name": "Cell infection and generation of conditioned medium",
"section_num": null
},
{
"section_content": "",
"section_name": "Syngeneic LLC tumor model, metastasis and drug administration",
"section_num": null
},
{
"section_content": "Syngeneic LLC or B16-F10 cells (5 × 10 5 suspended in PBS) were injected s.c.into the indicated mice (female or male mice aged 8-10 weeks).Fourteen days later, the tumors were harvested and analyzed further.Tumor formation and body weight were monitored every other day.The tumor volume was calculated as V = L × W 2 /2, where L and W are the length and width of the tumor, respectively. ",
"section_name": "Subcutaneous syngeneic mouse tumor model",
"section_num": null
},
{
"section_content": "One week after tamoxifen-induced EMCN knockout, luciferase reporter LLC or wild-type (1 × 10 6 suspended in PBS) tumor cells were injected into the mouse tail vein.Mice were randomly assigned to different experimental groups.After 21 days of tumor cell injection, the lung was dissected to observe the metastatic foci, and the tissue was fixed for subsequent section staining.For the neutrophil deletion experiment, mice were administered either anti-Ly6G + antibody (clone 1A8; Bio X Cell, 7.5 mg/kg) or control (PBS) once every three days in vivo. ",
"section_name": "Intravenous injection of lung metastasis mouse model",
"section_num": null
},
{
"section_content": "Syngeneic LLC tumor cells (3 × 10 5 or 5 × 10 5 suspended in PBS) were subcutaneously injected into the flanks of EMCN ecko and control mice 1 week after tamoxifen administration.Primary tumor growth was measured, and tumor growth was calculated.The primary tumor (approximately 1 cm in diameter) was surgically removed 15 or 21 days after implantation under anesthesia.The tissue was fixed for subsequent section staining, and the tumor was weighed.We observed lung metastasis 1 week, 2 weeks and 3 weeks after tumor resection by HE staining.After 21 days of primary tumor resection, LLCinjected mice showed significant distant lung metastasis.Therefore, 21 days after resection was selected as the end point of our follow-up experiments.For the therapy experiment, mice were administered either DAPT (GSI-IX, 20 mg/kg) or vehicle (DMSO:PEG400:Tween-80:NaCl = 10%:40%:5%:45%) once a day.The primary tumor was surgically removed with a diameter of 1 cm until the experimental end point criteria were reached.DAPT or vehicle was injected once every three days.For the combination treatment experiment, mice were administered either DAPT or vehicle once a day.Anti-TGF-β antibody (1D11; Bio X Cell 10 mg/kg) was injected once every three days.The primary tumor was surgically removed when a diameter of 1 cm was obtained or when the experimental endpoint criteria were reached.The investigators assessing endpoint criteria were blinded to the treatment administered.For survival experiments, humane endpoints included weight loss of 20% or greater, reduced activity, pale feet and visible symptoms of distress, such as hunching, closed eyes and isolation from cage mates. ",
"section_name": "Postsurgical metastasis model",
"section_num": null
},
{
"section_content": "To deplete Ly6G + neutrophils, mice were intraperitoneally injected with 7.5 mg/kg anti-Ly6G + (clone 1A8; BioXCell) once every three days.Neutrophil depletion was confirmed by immunofluorescence.For visualization of the luciferase reporter gene that is expressed by the LLC cells, d-Luciferin (PerkinElmer cat.#122796) was intraperitoneally injected (150 mg/kg), and the lungs were dissected 10 min after injection.Luciferase-positive regions were imaged using IVIS Lumina II (Caliper Life Sciences). ",
"section_name": "Depletion of neutrophils and in vivo imaging system (IVIS)",
"section_num": null
},
{
"section_content": "Tumor-bearing WT mice and EMCN ecko mice were injected intravenously with 100 µl of a mixture containing 2 mg/ml rhodamine-conjugated dextran (70 kDa) in PBS.After 30 min, the mice were euthanized, and cardiac perfusion was performed with 10 ml PBS.Lung tissue was fixed with 4% paraformaldehyde (PFA) at 4 °C for 24 h; washed in PBS for 5 min; passed through sucrose solutions of 10%, 20% and 30% for 24 h; and embedded in OCT.Frozen blocks were cut into 10-µm cryosections. Images were obtained using a Leica confocal microscope.The fluorescence intensity of 70 kDa rhodamine-dextran was quantified by using ImageJ software. ",
"section_name": "In vivo vessel permeability assay",
"section_num": null
},
{
"section_content": "Lung and tumor tissues were fixed in 10% formaldehyde solution in PBS at room temperature followed by routine dehydration, paraffin embedding, and tissue sectioning.Paraffin sections (4 µm) were stained with hematoxylin-eosin to observe the structure of the main organs and lung metastasis.For immunofluorescence staining, in brief, paraffin sections were dewaxed and placed into water.The sections were placed into sodium citrate buffer, heated in a microwave oven for antigen repair, restored to room temperature, and blocked with goat serum at room temperature for 1 h.Sections were incubated with the primary antibodies anti-Ly6G + (ab238132, 1:200), anti-Arg2 + (ab264071, 1:200) and anti-NOS2 (ab115819, 1:200) at 4 ℃ overnight.The slices were washed thrice with PBS for 3 min each time followed by incubation with Alexa Fluor 488-or 594-conjugated secondary antibodies (1:5000 dilutions) for 1 h.DAPI was used to stain the nucleus for 15 min, and samples were subject to imaging with a Leica fluorescence microscope.A routine protocol was performed for immunohistochemistry.The primary antibody dilutions were S100A8/ A9 (ab22506, 1:1000), anti-CD31 (D8V9E, 1:500), and anti-Ki67 (ab16667, 1:1000). ",
"section_name": "Immunofluorescence (IF) and immunohistochemistry (IHC)",
"section_num": null
},
{
"section_content": "Total RNA from cultured cells (HUVECs/shNCs and HUVECs/shEMCNs) and mouse lung tissues was isolated with TRIzol reagent (Invitrogen) as instructed.cDNA was synthesized from 2 μg of total RNA with random primers using a Thermo kit, and the concentration was measured by Colibri.mRNA expression was assessed based on the threshold cycle (Ct), and relative expression levels were calculated as 2 -ΔΔct after normalization to GAPDH expression.The primers used for quantitative real-time PCR are listed in Additional file 6: Table S1. ",
"section_name": "Quantitative real-time PCR",
"section_num": null
},
{
"section_content": "Western blot analysis was implemented using a standard protocol.The primary antibodies used for Western blot analysis included anti-endomucin (sc-65495, 1:500), antiendomucin (ab96315, 1:1000), anti-claudin-5 (ab131259, 1:1000), anti-MMP-9 (ab283575, 1:1000), anti-ZO-1 (ab216880, 1:1000), and anti-TGF-β (ab215715, 1:1000).The secondary antibodies included goat anti-mouse (ZB-2055, 1:10000), goat anti-rat (ZB-2040, 1:10000) and goat anti-rabbit (ZB-2306, 1:10000).Anti-β-actin (ab8226, 1:2000) was used as a loading control. ",
"section_name": "Western blotting",
"section_num": null
},
{
"section_content": "Total RNA was extracted using TRIzol reagent according to the manufacturer's protocol.RNA purity and quantification were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA).RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).Then, the libraries were constructed using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions.Transcriptome sequencing and analysis were performed by OE Biotech Co., Ltd.(Shanghai, China). ",
"section_name": "RNA isolation and library preparation",
"section_num": null
},
{
"section_content": "The libraries were sequenced on an Illumina HiSeq X Ten platform, and 150-bp paired-end reads were generated.Transcriptome sequencing and analysis were performed by OE Biotech (Shanghai, China).Raw data (raw reads) were processed using Trimmomatic [16].The reads containing poly-N and the low-quality reads were removed to obtain clean reads.Then, the clean reads were mapped to the reference genome using HISAT2 [17].The FPKM [18] value of each gene was calculated using cufflinks [19], and the read counts of each gene were obtained by htseq-count [20].DEGs were identified using the DESeq R package functions estimate SizeFactors and nbinomTest.A P value < 0.05 and fold change > 2 or fold change < 0.5 were set as the thresholds for significant differential expression.Hierarchical cluster analysis of DEGs was performed to explore gene expression patterns.GO enrichment and KEGG [21] pathway enrichment analyses of DEGs were performed using the R package based on the hypergeometric distribution.The sequencing coverage and quality statistics for each sample are summarized in Additional file 7: Table S2. ",
"section_name": "RNA sequencing and differentially expressed gene analysis",
"section_num": null
},
{
"section_content": "TCGA database provides high-throughput analysis of different tumors, including data on mRNA expression.Combining bioinformatics analysis with patient clinical information lays a foundation for improving cancer prevention and discovering new targets for treatment.In this study, we downloaded RNA-seq data of lung adenocarcinoma from TCGA database.mRNA data from 594 samples, including 59 normal samples and 535 lung cancer samples, and mRNA data from 1222 samples, including 112 normal breast samples and 1110 breast cancer samples, were used in this analysis.Using the R software package, the downloaded data were normalized and differentially analyzed to obtain differentially expressed mRNAs.We used the R package Survival to examine the prognostic potential of EMCN expression levels in cancers.Survival-relevant mRNAs with a log-rank P value < 0.05 were considered significant.The relevant data provided by TCGA are publicly available. ",
"section_name": "TCGA database",
"section_num": null
},
{
"section_content": "All graphs and statistical analyses were completed using GraphPad Prism software v8.0.Significant differences were evaluated using an independent sample T test, and multiple treatment groups were compared within individual experiments by ANOVA.TCGA data were downloaded from the cBioPortal website, and the logrank test was used for comparison of survival outcomes with the Kaplan-Meier method.Values of p < 0.05 were considered significant.All values are presented as the mean ± SD. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To identify the key genes and pathways involved in tumor metastasis, we compared the transcriptomic profiles of 55 normal and 538 lung cancer samples from TCGA database.In total, 3719 genes were significantly upregulated, and 1811 genes were significantly downregulated (Fig. 1A andB).Similar transcriptomic analysis of 112 normal 1110 breast cancer samples revealed upregulation of 7393 and downregulation of 3199 genes (Additional file 1: Fig S1A andS1B).Given that tumor cell interactions with endothelial cells play a critical role in cancer metastasis [22], we first focused on the expression analysis of these genes.We found that EMCN is specifically expressed in microvascular endothelial cells and showed remarkable downregulation in the majority of both metastatic lung and breast tumors.We hypothesized that EMCN downregulation may be important for tumor metastasis and recurrence (Fig. 1C and Additional file 1: Fig. S1C).To further investigate the clinical significance of EMCN expression in metastatic lung and breast cancer patients, we analyzed EMCN expression in patients with and without metastasis.The results showed that EMCN expression was significantly reduced in metastatic tumors (Fig. 1D and Additional file 1: Fig. S1D) and was strongly reduced in tumor recurrence (Fig. 1E and Additional file 1: Fig. S1E).These analyses revealed an important role of EMCN in the progression of tumor metastasis. ",
"section_name": "Downregulation of EMCN expression is related to metastasis and recurrence in tumor patients",
"section_num": null
},
{
"section_content": "We used an inducible knockout model using Tek-cre-ERT2 mice to drive endothelial-specific deletion of EMCN (hereafter referred to as EMCN ecko ).Endothelial EMCN inactivation was confirmed at the gene level by PCR and at the protein level by Western blot (Additional file 2: Fig. S2A andS2B).EMCN ecko mice appeared phenotypically normal; however, histological examination revealed an increase in red blood cells in the lungs of these animals compared with the wild-type (WT) mice (Additional file 2: Fig. S2C).To determine whether EMCN mediates tumor growth, syngeneic LLC cells were injected subcutaneously into the flanks of WT and EMCN ecko mice.Neoplasms were formed within 2 weeks and displayed no distinct impairment in growth rate or tumor angiogenesis between EMCN ecko and WT mice, which was also evidenced by Ki67 and CD31 staining, respectively (Fig. 2A-D).Consequently, EMCN expression in ECs does not affect tumor growth in vivo. ",
"section_name": "EMCN expression in endothelial cells does not affect tumor growth in vivo",
"section_num": null
},
{
"section_content": "Cancer cells penetrate blood vessels through intravasation and extravasation, and both processes require the ability to break through endothelial cells [23].Tumor cell metastasis typically occurs in small capillaries [24], and EMCN is a glycoprotein expressed in endothelial cells of these capillaries.We hypothesized that EMCN depletion in tumors may obliterate the integrity of the endothelial barrier and lead to an increase in cell infiltration.To determine the effect of EMCN deficiency on tumor metastasis in vivo (Fig. 2E), EMCN ecko mice were injected with LLC cells through the tail vein.The pathology results showed significantly higher metastasis in EMCN ecko mice (Fig. 2F), suggesting that EMCN depletion caused an increase in metastasis.Furthermore, EMCN knockout mice showed significantly reduced survival compared to WT mice when LLC cells were injected into the venous circulation (Fig. 2G). To construct a mouse model that is more consistent with the metastasis of clinical tumor patients, we resected subcutaneous tumors 2 weeks after implantation to simulate surgical resection in tumor patients and allowed time for disseminated tumor cells to form detectable metastases.The effect of EMCN knockout on lung metastasis was observed after resection of LLC primary tumors (Fig. 2H).EMCN ecko mice exhibited a significant increase in metastasis upon primary tumor resection compared with WT mice (Fig. 2I and Fig. S2D).We used a second animal model to confirm the same results, indicating that the occurrence of lung metastasis is not cell specific (Additional file 2: Fig. S2E).These results demonstrated that (i) loss of EMCN in the vascular endothelium results in markedly greater spontaneous metastases to the lung and (ii) the establishment of EMCN ecko model mice with greater spontaneous metastases. ",
"section_name": "EMCN deficiency in ECs aggravates spontaneous lung metastasis.",
"section_num": null
},
{
"section_content": "To further understand the mechanism of endothelial EMCN in tumor metastasis, we constructed a stable EMCN knockdown cell line in vitro.The results showed that EMCN knockdown significantly inhibited endothelial cell proliferation (Additional file 3: Fig. S3A andS3B). Next, we evaluated the gene expression profiles of EMCN (HUVEC/shEMCN) knockdown and control endothelial cells (HUVEC/Con313).Differentially expressed genes were selected as those showing either upregulation or downregulation by > twofold in HUVECs/shEMCN compared with HUVECs/Con313.KEGG analysis of differentially expressed genes showed that EMCN knockdown in HUVECs significantly affected cell junctions (Fig. 3A).Patricia A. D' Amore et al. reported that EMCN controls angiogenesis by regulating the activation of VEGFR2 in vitro [13].We further confirmed whether EMCN controls endothelial cell junctions in vitro.HUVECs/ Con313 and HUVECs/shEMCN were seeded on Matrigel to facilitate capillary tube formation.Strikingly, EMCN deficiency impeded HUVEC angiogenesis, which is consistent with previous studies [13] (Fig. 3B).In the in vivo study, we found that EMCN did not affect angiogenesis but did affect tumor metastasis.By comparing EMCN expression levels in naive, premetastatic and metastatic lung tissue, we found that EMCN expression levels were decreased significantly in tumor metastasis (Additional file 3: Fig. S3C).Thus, we hypothesized that endothelial EMCN deficiency changes vascular barrier properties, such as vascular permeability.Thus, we established a pulmonary microvascular permeability model by intravenous injection of rhodamine-dextran (70 kDa).After 30 min, the permeability of high-molecular-weight rhodamine in the lung parenchyma was analyzed by fluorescence microscopy.The results revealed slight leakage in the lungs of EMCN ecko mice compared with WT mice (Additional file 3: Fig. S3D).Interestingly, EMCN ecko tumor-bearing mice exhibited significantly increased lung vascular permeability compared with WT mice, as indicated by the fluorescence signal intensity observed in frozen lung sections (Fig. 3C,D).The intercellular junction of endothelial cells composed of adhesive junctions and tight junctions is the key factor in maintaining vascular integrity.ZO-1 and Claudin5 are important adhesion molecules that maintain endothelial cell-cell contact [25][26][27].To explore how EMCN regulates vascular permeability in vivo, we further detected changes in ZO-1 and Claudin5 in the lungs of WT and EMCN ecko tumorbearing mice.The results showed that EMCN deficiency caused a significant reduction in ZO-1 and Claudin5 expression (Fig. 3E).In brief, these results suggested that EMCN depletion affected the expression of ZO-1 and Claudin5 and the integrity of the vascular barrier of pulmonary capillaries, causing an increase in vascular permeability. ",
"section_name": "EMCN deficiency affects gene expression profiles associated with cell junctions and vascular permeability",
"section_num": null
},
{
"section_content": "Next, we attempted to clarify the steps of the metastatic cascade affected by EMCN defects.Based on our results indicating (i) no difference in primary tumor growth between WT and EMCN ecko mice (Fig. 2B) and that (ii) EMCN deficiency increased lung metastasis (Fig. 2F andI) and pulmonary vascular permeability (Fig. 3D), we hypothesized that EMCN deletion might contribute to enhanced colonization of metastatic cells after changes in the host microenvironment, which relies on the formation of a lung metastatic niche.Vascular permeability is considered to be one of the important characteristics of the premetastatic niche [28] that plays an important role in tumor metastasis [29][30][31].Transcriptome sequencing and protein detection were used to evaluate whether EMCN defects in endothelial cells could affect the formation of pulmonary premetastatic niches by affecting vascular permeability.MMP9, TGF-β and S100A8/ A9 have been demonstrated to increase lung inflammation or immunosuppressive factors and play a key role in the initiation of the premetastatic niche [28,32].Next, transcriptome analysis of lung tissue was performed in EMCN ecko mice, WT mice and tumor-bearing WT mice.We focused on the changes in premetastatic nicherelated genes.Differentially expressed genes were identified as those upregulated or downregulated by > 1.5-fold in the groups of tumor-bearing WT mice vs. WT mice and EMCN ecko vs. WT mice.We found that EMCN deficiency led to changes in the premetastatic niche in the lung, which was similar to the premetastatic niche induced by LLC tumors (Fig. 4A).Furthermore, EMCN deficiency was associated with changes affecting the host microenvironment, resulting in an increase in genes related to the premetastatic niche (TGFB, S100A8/A9, MMPs, etc.).The key factors in the premetastatic niche were detected, and the expression of S100A8/A9, MMP9 and TGF-β was increased in the lungs of EMCN ecko mice. The expression levels of MMP9, TGF-β and S100A8/A9 were confirmed by Western blot and IHC, respectively (Fig. 4B-D).In brief, these results indicated that EMCN deficiency leads to the formation of a lung metastatic niche. ",
"section_name": "EMCN deficiency leads to the formation of a lung premetastatic niche by upregulating MMP9, TGF-β and S100A8/A9",
"section_num": null
},
{
"section_content": "It has been demonstrated that overexpression of EMCN can prevent neutrophil-endothelial cell interactions in vitro and inhibit the infiltration of CD45 + and NIMPR14 + (neutrophil marker) cells, but there were no changes in F4/80 + (monocyte/macrophage marker) cells [12].An increasing number of studies have confirmed that neutrophils play an important role in the formation of a premetastatic niche in tumor lung metastasis [33,34].We analyzed the premetastatic niche lungs of WT and EMCN ecko tumor-bearing mice by RNA-seq.The results showed that EMCN deletion affected the inflammatory response and neutrophil chemotaxis (Fig. 4E).The increase in cytokines was measured by qRT-PCR (Fig. 4F).Therefore, we hypothesized that EMCN deficiency may lead to the aggregation of neutrophils in the lung, promote the formation of a premetastatic niche, and thereby promote growth and metastasis.We next detected neutrophil infiltration in the premetastatic niche and metastatic lung of WT and EMCN ecko mice by immunofluorescence staining and found that Ly6G + and S100A9 + (neutrophil marker) cells were significantly recruited in the lungs of EMCN ecko tumor-bearing mice compared with WT tumor-bearing mice (Fig. 4G, Additional file 3: Fig. S3E).The above results led to the conclusion that EMCN deletion can cause neutrophil infiltration in vivo. ",
"section_name": "EMCN deficiency promotes the recruitment of neutrophils to lung metastatic foci",
"section_num": null
},
{
"section_content": "Next, we evaluated whether the increased neutrophils caused by EMCN deficiency are functional, revealing an important mechanism of lung metastasis.Neutrophils in vivo were depleted with Ly6G + antibody before and after the tail vein injection of LLC cells into EMCN ecko mice.Through the formation of lung metastasis foci and in vivo imaging, we found that neutrophil depletion reduced lung metastasis in vivo (Fig. 5A andB).In brief, these results demonstrated that Ly6G + neutrophils play an important role in lung metastasis caused by EMCN deficiency. ",
"section_name": "EMCN deficiency promotes lung metastasis in a neutrophil-dependent manner",
"section_num": null
},
{
"section_content": "A deeper understanding of the metastasis process is needed to develop new therapies for cancer patients.We assessed whether the increased TGF-β was functionally important in LLC lung metastasis of EMCN ecko mice.It has been reported that the polarization of N2 neutrophils promotes tumor metastasis of breast and gastric cancers [35,36].TGF-β has been shown to play an important role in inducing N1 to N2 polarization [37].Through bioinformatics analysis of TCGA dataset and experimental verification, we found a negative correlation between EMCN and TGF-β (Figs. 4C and5C).Therefore, we hypothesized that EMCN deletion led to an increase in TGF-β in the microenvironment, promoted the polarization of N1 neutrophils into N2 neutrophils, and thereby promoted lung metastasis.Therefore, specific blockade of TGF-β-induced neutrophil polarization is a meaningful therapeutic strategy to treat lung metastasis. To further assess the anti-TGF-β suppressive effect of neutrophil polarization, we injected LLC cells subcutaneously into WT and EMCN ecko mice.EMCN ecko mice were treated with or without anti-TGF-β antibody and examined for N1-/N2-associated markers.Consistent with the above-described results (Fig. 2B), EMCN deletion did not affect growth but promoted lung metastasis of primary tumors in the WT and EMCN ecko groups (Fig. 5G andH).However, compared with EMCN ecko mice, anti-TGF-β antibody did not affect tumor growth but inhibited lung metastasis caused by EMCN deletion (Fig. 5E-H and Additional file 3: Fig S3F).We also examined the expression of relevant N1-and N2-neutrophil markers in lung metastatic tumors by immunofluorescence analysis and found that EMCN ecko mice could induce N2 neutrophils (Arg2 + ) to infiltrate metastatic tumors.However, anti-TGF-β antibody significantly inhibited N2 neutrophils, and N1 neutrophil infiltration was primarily observed (Fig. 5I and Additional file 3: Fig S3G ).Taken together, these results strongly suggest that the anti-TGF-β antibody is a promising therapeutic target that can be used to prevent or treat lung metastasis caused by neutrophil polarization. ",
"section_name": "Anti-TGF-β suppresses lung metastasis by blocking TGF-β-induced N2 neutrophil polarization",
"section_num": null
},
{
"section_content": "Previous studies have identified the key regulatory relationship of the Notch signaling pathway in the regulation of EMCN expression in hepatic ischemia/reperfusion injury.Pharmaceutical Notch blockade dramatically upregulated EMCN and prevented trans-endothelial migration of neutrophils in vitro.Furthermore, in RBPj (integrated transcription factor of typical Notch signaling) knockout mice, the Notch signal was inactivated, but the expression of EMCN was upregulated [15].Through bioinformatics analysis of TCGA data, an obvious negative correlation was noted between EMCN and Notch1 (Fig. 6A).To further investigate whether Notch regulates EMCN expression, EMCN protein expression was detected by inhibiting NICD (a key protein of the Notch signaling pathway).The results showed that NICD inhibition significantly increased the expression level of EMCN in HUVECs (Fig. 6B and Additional file 4: Fig. S4A).After we cocultured HUVECs with different tumor cell-conditioned media, the results showed that tumor cells significantly activated NICD in HUVECs, which was consistent with previous studies [9].Interestingly, NICD activation significantly inhibited the expression of EMCN on HUVECs (Fig. 6C and Additional file 4: Fig. ",
"section_name": "Notch inhibitor suppresses tumor growth and metastasis by upregulating EMCN",
"section_num": null
},
{
"section_content": "). Accordingly, we used the Notch pathway inhibitor DAPT, a γ-secretase inhibitor, to detect its effect on tumor growth and metastasis using a syngeneic mouse model.To determine whether DAPT can inhibit tumor growth and metastasis, we treated tumor-bearing mice with DAPT or vehicle.The results showed that DAPT slightly inhibited tumor growth and metastasis compared with the vehicle group (Fig. 6D-F).Immunohistochemistry results for Ki67 staining confirmed these findings (Additional file 4: Fig. S4C and4D).Western blot analysis showed that EMCN expression was significantly upregulated in the lungs of DAPT-treated mice compared with that in the vehicle-treated mice (Additional file 4: Fig. S4E).These results confirmed that DAPT could inhibit tumor growth and elicit a suppressive effect on lung metastases by upregulating EMCN. ",
"section_name": "S4B",
"section_num": null
},
{
"section_content": "TGF-β weakens the intrinsic antitumor potential of immune cells in the tumor microenvironment by increasing the inhibitory effect on key immune cells of innate and adaptive immunity.Therefore, the antitumor response of myeloid cells and lymphocytes is hypothesized to be enhanced by inhibiting TGF-β [38].It is worth mentioning that due to the limited clinical efficacy of TGF-β inhibitor monotherapy, a large number of combined therapies are being assessed, such as combined cytotoxic drugs, radiotherapy, and immune checkpoint inhibitors [39][40][41][42].In light of the data described above, DAPT has a good therapeutic effect on tumor growth and metastasis in syngeneic mice, whereas the use of DAPT as a Notch signaling pathway inhibitor in combination with anti-TGF-β antibody has not been reported.We again used the mouse tumor metastasis model of syngeneic subcutaneous tumor grafting and surgical resection.Once tumors are palpable, the animals were assigned to different treatment systems as shown in Fig. 6D.Combined DAPT and anti-TGF-β administration did not significantly affect mouse body weight or damage to important organs compared with control mice, indicating that the regimen with a low dose of DAPT and anti-TGF-β is relatively safe (Additional file 5: Fig. S5A andB).The growth of subcutaneous tumors was significantly inhibited (Fig. 6D andE).Immunohistochemical analysis of Ki67 revealed differences in proliferation (Additional file 4: Fig. S4C).Tumor metastasis was found in the lung metastasis foci and HE-stained sections (Fig. 6F, Additional file 4: Fig. S4D).Notably, we found that the combination of DAPT and anti-TGF-β antibody dramatically reduced the area of the metastatic nodules and slightly inhibited tumor growth compared to the DAPT group (Fig. 6F).We used a second animal model to confirm the same results (Additional file 5: Fig. S5C andD).The survival rate of mice was significantly improved (Additional file 5: Fig. S5E).Taken together, we demonstrated the important role of DAPT combined with anti-TGF-β antibody in tumor growth and metastasis, especially the significant inhibition of tumor metastasis and improved survival using a syngeneic mouse model. ",
"section_name": "Combined treatment with a Notch inhibitor and an anti-TGF-β antibody synergistically reduces tumor growth and metastasis",
"section_num": null
},
{
"section_content": "To further validate our experimental results, we examined the clinical significance of Notch1 and EMCN in lung cancer by analyzing TCGA dataset.We evaluated the relationship between Notch1 and EMCN expression levels and patient survival.The results showed that patients with high EMCN expression exhibited a higher survival rate, whereas patients with high Notch1 expression exhibited a significantly lower survival rate (Additional file 5: Fig. S5F). In conclusion, we demonstrate a new antimetastatic effect of EMCN on lung metastasis.EMCN deletion caused a remarkable increase in metastasis by affecting the formation of the premetastatic niche.TGF-β in the premetastatic niche promoted the polarization of neutrophils and exacerbated lung metastasis.Pharmacological inhibition of Notch improved EMCN expression, inhibited tumor metastasis, and showed additive effects when combined with anti-TGF-β antibody therapy.Taken together, we demonstrate a possible new therapeutic strategy involving Notch inhibitors and anti-TGF-β antibodies in clinical tumor patients (Fig. 7). ",
"section_name": "The clinical significance of EMCN and Notch expression in cancer patients from TCGA",
"section_num": null
},
{
"section_content": "Our data from LLC primary tumors between WT and EMCN ecko mice revealed no differences in primary tumor growth and blood vessel formation.This finding led us to study the metastasis process downstream of the primary tumor and alterations in vascular function.We found higher EMCN expression levels in the lung compared with other tissues, including the heart, liver and spleen.We explored the hypothesis that EMCN knockout mice had increased lung colonization following tail vein injection of LLC cells compared to wild-type mice in part due to increased extravasation and increased permeability of pulmonary vessels, which subsequently leads to alterations in the premetastatic niche.These results provide insight into how EMCN expression in endothelial cells affects tumor metastasis. However, identifying specific EMCN downstream molecular changes that are critical for metastasis can be very difficult.This challenge becomes further exacerbated when we consider that EMCN is not expressed on tumor cells but on normal endocytic endothelial cells.It has been found that the combination of the tumor cell surface ligand app and endothelial cell surface receptor DR6 can lead to programmed death of endothelial cells and promote tumor metastasis [22].Therefore, EMCN on endothelial cells may interact with ligands on various stroma and tumor cells.However, previous studies have found that EMCN, as a member of the mucin family, is a sialylated glycoprotein and has more anti-adhesion properties [43].To date, no ligands that bind to EMCN have been identified.Many studies have demonstrated the role of EMCN in endothelial cell angiogenesis [44].Although we did not observe significant differences in angiogenesis in vivo, this does not exclude the role of EMCN in the vascular function of endothelial cell formation.Few studies have found that molecules on endothelial cells can affect endothelial permeability and tumor metastasis [45].Therefore, we demonstrated the important role of EMCN deletion in endothelial cell infiltration and tumor premetastatic niche formation. Lung metastasis is commonly observed in different types of cancer, including breast cancer, gastrointestinal tumors, melanoma and different types of sarcomas in addition to lung cancer itself [46].In lung metastasis of tumors, TGF-β can induce the secretion of ANGPTL4.These secreted mediators enhance the extravasation of tumor cells in the lung by weakening the cell-cell connection between endothelial cells [47].We found changes in TGF-β levels in the lungs of WT and EMCN ecko tumor-bearing mice.TGF-β is critical for immunosuppression in the tumor microenvironment.It inhibits the function of many components of the immune system and promotes tumor occurrence.Recent studies have shown that TGF-β plays a role in tumor immune escape and adverse responses to tumor immunotherapy [48,49].The unique function and regulation of neutrophils in cancer are closely related to the formation of lung premetastatic niches in tumor-bearing mice [50].An interesting difference we observed was the increase in neutrophils in EMCN ecko premetastatic lung and tumor-bearing lung.Because EMCN has been shown to prevent leukocytes from adhering to endothelial cells [12], EMCN deletion may affect neutrophil recruitment to the lung tissue.Neutrophils in the lung play a role in forming a premetastatic niche and promoting tumor metastasis after polarization [36].We demonstrated that lung neutrophils lacking EMCN were largely increased and induced to form N2 neutrophils by TGF-β in the microenvironment to promote tumor metastasis and growth.After neutralizing TGF-β by injecting a TGF-β antibody in vivo, the Fig. 7 A proposed working model.The loss of endothelial EMCN caused an increase in vascular permeability, produced a premetastatic niche and promoted tumor lung metastasis.Downregulation of EMCN expression was reversed by treatment with a Notch inhibitor combined with an anti-TGF-β antibody to inhibit lung metastasis inhibitory effect of the N2 neutrophil phenotype was observed. Although our research focuses on primary tumors and lung metastasis, there are additional problems that need to be discussed.Compared with WT, the lung lacking EMCN may or may not be the only organ that provides a favorable premetastatic niche for tumor cell colonization.For example, whether EMCN defects in the liver provide a favorable premetastatic niche for the liver metastasis of mouse colorectal cancer cells warrants further study using a liver metastasis model of colorectal cancer.We confirmed that EMCN affects vascular function, but we did not further study the other biological functions of EMCN on endothelial cells, such as endothelial cell senescence and programmed death. Preclinical studies have shown that TGF-β inhibition combined with checkpoint inhibitors can significantly enhance its immune effect, whereas TGF-β inhibition may show limited efficacy as a monotherapy [51,52].In another combined application study, radiotherapy combined with blocking TGF-β antibody enhanced the systemic antitumor response [53].A series of Notch signaling pathway inhibitors have been tested in phase I/ II clinical trials of various types of cancer, and the complexity of notch inhibition and alternative carcinogenic signals that may be provided by other pathways have produced more adverse reactions [54].Based on our experimental data, the growth and metastasis of mouse syngeneic tumors was inhibited by TGF-β antibody combined with DAPT.However, bioinformatics analysis of limited clinical data showed that although the difference was not highly significant, the survival rate of patients with high expression of EMCN and low expression of Notch was significantly increased.Our combined therapy research in mice provides a new strategy for the clinical prevention and treatment of tumor metastasis. In conclusion, we demonstrate that EMCN deficiency in endothelial cells promotes metastasis by providing a suitable premetastatic niche for cancer cell extravasation and lung colonization.Therefore, targeting notchmediated upregulation of EMCN in endothelial cells combined with TGF-β inhibition may represent a new method to prevent or treat metastasis.Future extensive studies should determine the value of EMCN expression as a potential prognostic biomarker in patients with melanoma and other highly metastatic cancers. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "We thank Sunil C Kaul and Renu Wadhwa for Writing-Reviewing and Editing. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This work was supported by the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2021-I2M-1-013, 2016-I2M-3-019) and the National Key Research and Development Program of China (Grant No. 2021YFF0702800).This study received funding support for this work from the Special Research Fund for Central Universities, Peking Union Medical College (No. 3332020050). ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "Only publicly available data were used in this study, and the data sources and handling of these data are described in the Materials and Methods.Further information is available from the corresponding author upon request. ",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "CCK-8: Cell counting kit-8; DAPT: γ-Secretase inhibitor; DEGs: Differentially expressed genes; EMCN: Endomucin; EMCN ecko : Endothelial cell knockout EMCN; HUVEC: Human umbilical vein endothelial cells; IF: Immunofluorescence; IHC: Immunohistochemistry; MMP-9: Matrix metalloproteinase 9; NICD: Notch intracellular fragment; qRT-PCR: Quantitative reverse transcriptionpolymerase chain reaction; S100A8/A9: S100 calcium-binding protein A8/A9; TCGA : The Cancer Genome Atlas; TGF-β: Transforming growth factor-β; WT: Wide type; ZO-1: Zonula occludens 1. The online version contains supplementary material available at https:// doi.org/ 10. 1186/ s12967-022-03649-4.Additional file 6: Table 1.Sequences for primers for qRT-PCR and target sequences of shRNA. RG and GZ performed conceptualization, methodology, formal analysis and writing-original draft preparation.DZ performed data curation and visualization.WZ performed visualization and supervision.XY performed validation.ML was responsible for gathering resources.RG and WZ was responsible for funding acquisition.The work reported in the paper has been performed by the authors, unless clearly specified in the text.All authors read and approved the final manuscript. The animal study was reviewed and approved by the Animal Experiments Committee of the Chinese Academy of Medical Sciences (IACUC: GR21003). Not applicable. The authors declare no conflict of interest. • 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": "Abbreviations",
"section_num": null
},
{
"section_content": "CCK-8: Cell counting kit-8; DAPT: γ-Secretase inhibitor; DEGs: Differentially expressed genes; EMCN: Endomucin; EMCN ecko : Endothelial cell knockout EMCN; HUVEC: Human umbilical vein endothelial cells; IF: Immunofluorescence; IHC: Immunohistochemistry; MMP-9: Matrix metalloproteinase 9; NICD: Notch intracellular fragment; qRT-PCR: Quantitative reverse transcriptionpolymerase chain reaction; S100A8/A9: S100 calcium-binding protein A8/A9; TCGA : The Cancer Genome Atlas; TGF-β: Transforming growth factor-β; WT: Wide type; ZO-1: Zonula occludens 1. ",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https:// doi.org/ 10. 1186/ s12967-022-03649-4.Additional file 6: Table 1.Sequences for primers for qRT-PCR and target sequences of shRNA. ",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "",
"section_name": "Additional",
"section_num": null
},
{
"section_content": "RG and GZ performed conceptualization, methodology, formal analysis and writing-original draft preparation.DZ performed data curation and visualization.WZ performed visualization and supervision.XY performed validation.ML was responsible for gathering resources.RG and WZ was responsible for funding acquisition.The work reported in the paper has been performed by the authors, unless clearly specified in the text.All authors read and approved the final manuscript. ",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "The animal study was reviewed and approved by the Animal Experiments Committee of the Chinese Academy of Medical Sciences (IACUC: GR21003). ",
"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 no conflict of interest. • 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
}
] |
10.3390/curroncol30100657
|
Patient Derived Xenografts (PDX) Models as an Avatar to Assess Personalized Therapy Options in Uveal Melanoma: A Feasibility Study
|
<jats:p>Uveal melanoma is the most common primary intraocular malignancy in adults. Up to 50% of UM patients develop metastatic disease, usually in the liver. When metastatic, the prognosis is poor, and few treatment options exist. Here, we investigated the feasibility of establishing patient-derived xenografts (PDXs) from a patient’s tumor in order to screen for therapies that the patient could benefit from. Samples obtained from 29 primary tumors and liver metastases of uveal melanoma were grafted into SCID mice. PDX models were successfully established for 35% of primary patient tumors and 67% of liver metastases. The tumor take rate was proportional to the risk of metastases. PDXs showed the same morphology, the same GNAQ/11, BAP1, and SF3B1 mutations, and the same chromosome 3 and 8q status as the corresponding patient samples. Six PDX models were challenged with two compounds for 4 weeks. We show that, for 31% of patients with high or intermediate risk of metastasis, the timing to obtain efficacy results on PDX models derived from their primary tumors was compatible with the selection of the therapy to treat the patient after relapse. PDXs could thus be a valid tool (“avatar”) to select the best personalized therapy for one third of patients that are most at risk of relapse.</jats:p>
|
[
{
"section_content": "Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, with an incidence of 5-8 per million individuals per year in Caucasian populations [1].Despite successful treatment of the primary tumor by either surgery and/or radiotherapy, up to 50% of UM patients develop metastatic disease, usually in the liver, even several years after the primary treatment [2,3].The median interval between primary tumor and metastasis was 68 months (range 19-81) in our series [4].Tools based on gene expression profiling (Decision Dx-UM [5]) or on genetic aberrations [6] allow us to estimate the risk of metastasis and to adapt surveillance.After a diagnosis of liver metastases, the median overall survival is around 15 months [3,7,8], and treatment options are limited at this stage.Although a small fraction (<5%) of patients show an unexpected response to treatment, the short survival time of most patients does not generally allow testing several lines of subsequent treatments.Therefore, tumor models derived from each individual patient would allow us to test different treatment options in parallel and selecting the most efficient one(s) for the patient.Patient-derived xenografts (PDXs) are in vivo models which are based on the graft of human tumor fragments in immunocompromised mice [9][10][11][12].These xenografts retain the histopathological and genetic features of the original patients' cancers, making them a valid tool to expand primary tumors, predict cancer response to therapy, and determine new therapeutic biomarkers [13][14][15][16][17][18].PDX mice models can be used as an avatar of the original tumor to test several drugs or a combination of drugs simultaneously and select the best therapy for their corresponding patients.The therapeutic benefits of avatar mice have been demonstrated by several groups in colon cancer, lung cancer, pancreatic cancer, renal carcinoma, and breast cancer [10,[17][18][19][20][21][22][23].Moreover, PDX models have been used as tools for clinical trials and co-clinical trials [12]. In UM, these avatar models could be of particular interest, since primary tumors and metastases tend to be highly similar, at least from a genomic point of view [24].These models could thus potentially be developed from the primary tumor material in anticipation of metastatic recurrence and allow for the rapid testing of multiple drugs. The present study is a proof-of-concept study evaluating the feasibility of avatar models to predict individual therapy responses in UM.We evaluated whether the time required to obtain the PDXs from the primary tumor, and to challenge them with different treatments, is compatible with the short life expectancy of the patients. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "A total of 30 patient samples were obtained from surgical left-over.All patients had previously given their informed consent for experimental research on residual tumor tissues.One of the samples was not tumoral and was excluded.Among the remaining 29 samples, 20 were obtained from primary UM and 9 from liver metastases.Table 1 summarizes the characteristics of patients.Twelve samples came from men and seventeen from women.Age ranged from 30 to 83 years old in both men and women.After surgery, some tumor specimens were fixed for further morphological and histological analyses, and others were frozen for further genomic analyses. ",
"section_name": "Patients and Tumor Samples",
"section_num": "2.1."
},
{
"section_content": "Fresh tumor samples obtained from pathologists were transplanted into the interscapular fat pad of two to four immunodeficient female SCID mice (Janvier Labs, Paris, France), 5 to 7 weeks old, under total xylazine/ketamine anesthesia (=passage 0).Mice were maintained in specific pathogen-free animal housing (Institut Curie, Paris, France) and regularly observed for tumor growth.Animal care and use for this study were performed following the recommendations of the European Community (2010/63/UE) for the care and use of laboratory animals and under the supervision of authorized investigators (APAFIS#25870-2020060410487032 v1).Mice were weighed and tumors were measured at least once a week.Tumor volumes (V), calculated by measuring two perpendicular diam-eters with calipers, were calculated according to the formula V = a × b 2 /2, where a and b are the largest and smallest perpendicular tumor diameters.At a volume of ~800 mm 3 , tumors were removed, and specimens were frozen directly in liquid nitrogen for molecular analysis, or in a DMSO-FCS solution for cryopreservation and expansion.Tumors were also fixed in mixed formalin/acetic acid for histological analysis.The xenografts were concomitantly evaluated for their response to 2 chosen treatments.Each tumor was subsequently transplanted into 20 immunodeficient SCID mice for therapeutic assessment. ",
"section_name": "Establishment of Uveal Melanoma Xenografts",
"section_num": "2.2."
},
{
"section_content": "For in vivo experiments, everolimus (Certican, Novartis, Basel, Switzerland) was suspended in glucose 5% and administered orally at 2 mg/kg, 5 days/week, for 4 weeks.Dacarbazin (Deticene, Sanofi Aventis, Paris, France) was administered intraperitoneally at 40 mg/kg, for 5 consecutive days.When converted to human equivalents, these doses are similar to those received by patients in the clinic.For in vivo therapeutic studies, 20 female SCID mice (Janvier Labs, Paris, France) were xenografted with a tumor fragment of 20-30 mm 3 (Figure 1).Mice bearing growing tumors with a volume of 60-200 mm 3 were randomly assigned to the control or treatment groups (number of animals per group is detailed in the figure legends).Animals with tumor volumes outside this range were excluded.Treatments were started on day 1. Mice were weighted and tumors measured at least once a week.Xenografted mice were sacrificed after 4 weeks of treatment or when at least one tumor reached a volume of 2500 mm 3 .Relative tumor volumes (RTV) were calculated from the following formula: RTV = (Vx/V1), where Vx is the tumor volume on day x and V1 is the tumor volume at the initiation of therapy (day 1).Growth curves were obtained by plotting the mean values of RTV on the Y axis against time on the X axis, expressed as days after the start of treatment.Antitumor activity was evaluated according to tumor growth inhibition (TGI), calculated according to the formula percent GI = 100 -(RTVt/RTVc × 100), where RTVt is the mean RTV of treated mice and RTVc is the mean RTV of controls, both at a given time point when the antitumor effect was optimal.Fifty percent TGI or more was considered as a meaningful biological effect.Statistical significance of differences observed between the individual RTVs corresponding to the treated mice and control groups was calculated using the two-tailed Mann-Whitney test. ",
"section_name": "In Vivo Therapeutic Assessment",
"section_num": "2.3."
},
{
"section_content": "The morphology of each xenograft was compared with the histological findings of the corresponding patient's tumor.For light microscopy examination, 4 µm thick AFA-fixed paraffin-embedded sections were stained with H&E Safran.Mice were weighted and tumors measured at least once a week.Xenografted mice were sacrificed after 4 weeks of treatment or when at least one tumor reached a volume of 2500 mm 3 .Relative tumor volumes (RTV) were calculated from the following formula: RTV = (Vx/V1), where Vx is the tumor volume on day x and V1 is the tumor volume at the initiation of therapy (day 1).Growth curves were obtained by plotting the mean values of RTV on the Y axis against time on the X axis, expressed as days after the start of treatment.Antitumor activity was evaluated according to tumor growth inhibition (TGI), calculated according to the formula percent GI = 100 -(RTVt/RTVc × 100), where RTVt is the mean RTV of treated mice and RTVc is the mean RTV of controls, both at a given time point when the antitumor effect was optimal.Fifty percent TGI or more was considered as a meaningful biological effect.Statistical significance of differences observed between the individual RTVs corresponding to the treated mice and control groups was calculated using the two-tailed Mann-Whitney test. ",
"section_name": "Histopathological Analyses",
"section_num": "2.4."
},
{
"section_content": "The morphology of each xenograft was compared with the histological findings of the corresponding patient's tumor.For light microscopy examination, 4 µm thick AFAfixed paraffin-embedded sections were stained with H&E Safran. ",
"section_name": "Histopathological Analyses",
"section_num": "2.4."
},
{
"section_content": "Frozen tumor (PDX and Patient tumor) samples were used for DNA extraction using the Qiagen ® (Venlo, The Netherlands) kit after the evaluation of tumor cell content on a frozen section.Samples containing > 50% of tumor cells were initially considered suitable for DNA extractions and genomic analyses.The extracted tumor DNA was used for Once the target and corresponding drug had been identified, it was administered to the mice (D).The most effective drug would be administered to the patient. ",
"section_name": "Establishment of the Tumor Molecular Profile",
"section_num": "2.5."
},
{
"section_content": "Frozen tumor (PDX and Patient tumor) samples were used for DNA extraction using the Qiagen ® (Venlo, The Netherlands) kit after the evaluation of tumor cell content on a frozen section.Samples containing > 50% of tumor cells were initially considered suitable for DNA extractions and genomic analyses.The extracted tumor DNA was used for mutations and gene copy number analyses as described in [25].For targeted sequencing, between 10 and 50 ng of DNA was used to prepare indexed paired-end libraries using the Illumina TruSeq Custom Amplicon Low Input (TSCA-li) kit, spanning 6 genes of interest (GNAQ, GNA11, CYSLTR2, EIF1AX, BAP1 and SF3B1) with 1536 amplicons distributed along 21 chromosomes.For CGH, 700 to 1000 ng of tumor DNA and reference DNA were labeled, purified, and cohybridized in equal quantity to NimbleGen arrays or Agilent microarrays.Nimblegen arrays were scanned with a GenePix 4000B scanner using GenePix software V.6.6 (Molecular Devices, San Jose, CA, USA), and data were extracted using Nim-bleScan software V.2.5.Files produced by Nimblescan software were then analyzed with SignalMap V.1.9.For Agilent, images were acquired with a SureScan microarray scanner using CytoScan software V.2.7, then analyzed with CytoGenomics software V.3.0.2.11. Selected gene copy number alterations were assessed using Cytoscan HD according to the manufacturer's protocol (Affymetrix ® , Santa Clara, CA, USA).Two hundred and fifty ng of genomic DNA was employed to conduct the target preparation and hybridized microarrays.When the amount of available genomic DNA was below 250 ng, a first whole genome amplification (Qiagen, REPLI-g Mini Kit PN:150023) step was implemented to the assay.Negative and positive controls were added to all batches of samples to ensure the quality control of analyses (Affymetrix normal DNA).Hotspot detection was performed by targeted sequencing using the Ion Ampliseq cancer panel V1.The protocols were detailed in [25]. ",
"section_name": "Establishment of the Tumor Molecular Profile",
"section_num": "2.5."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "We here aim to evaluate the feasibility of creating avatar PDX models of UM to assess the responses to (targeted) therapy in these models within a timeframe that is compatible with the evolution of this disease in patients.The different steps to obtain a valuable tool for personalized medicine are shown in Figure 1.A total of twenty-nine UM patient samples were obtained after surgery (Figure 1A), among which twenty were from primary UM and nine from liver metastases.Table 1 summarizes the characteristics of the patients.The tumor samples were grafted onto mice (Figure 1B) and once a PDX had grown (passage 0), we fixed one fragment of PDX for histology and froze one fragment to determine the genomic profile of the PDX (Figure 1C').In parallel, several fragments were frozen to secure the possibility of testing the efficacy of new drugs in the future.The next step was the expansion of the obtained PDXs into 20 new mice (Figure 1C) and, concomitantly, the molecular analysis of PDXs and patients (Figure 1C').The comparison of PDXs to the corresponding patient's tumor was used to select compounds for drug screening.Genomic analyses were conducted as described previously [25,26] and results were provided within 2 weeks.The last step is to assess the antitumor efficacy of selected compounds in the mice bearing the xenografts (Figure 1D) to choose the best treatment for the corresponding patient. ",
"section_name": "Study Design",
"section_num": "3.1."
},
{
"section_content": "We first evaluated the tumor take rate.Tumor take was considered successful when the volume of the PDX tumor reached at least 60 mm 3 .The engraftment was successful for 7/20 (35%) of primary tumors and 6/9 (67%) of metastatic samples (Table 2).There was a tendency for a higher tumor take rate in xenografts obtained from metastases (67%) than those from primary tumors (35%) (p = 0.2254).The overall tumor take rate was 45%. ",
"section_name": "Establishment of PDX",
"section_num": "3.2."
},
{
"section_content": "Copy number alterations of chromosomes 3 and 8 frequently occur in UM and predict metastatic relapse in patients [6].We determined the genomic risk in patients' tumor samples and compared this with the tumor take rate in mice (Table 2).High risk (HR) of metastases was defined by the loss of chromosome 3 (chr3) and gain of chromosome 8q in patients [6,27].The tumor take was around 50% in high-risk patients whose primary tumors displayed both loss of chr3 and gain of 8q (Table 2).In patients with an intermediate risk (IR, either loss of chr3 or gain of chr 8q in the primary tumor), the tumor take rate was 30%.In four patients, chromosome 3 and 8 status was normal.Only one PDX was obtained from these samples, but it stopped growing at passage 1. In the metastatic patient samples, the take rate was 100% for high-risk tumors, 60% for intermediate-risk, and 0% for low-risk.We thus observed a correlation between tumor take rate and genomic risk as determined by patients' tumors.One PDX belonging to the high-risk group stopped growing at passage 1.We decided to set aside this tumor for further investigations.The status of chromosome 3 and chromosome 8 and the risk of TTR: Tumor take rate; * 1 tumor stopped growing at passage 1. ",
"section_name": "Genomic Risk of the Patient Correlates with Tumor Take Rate in Mice",
"section_num": "3.3."
},
{
"section_content": "Copy number alterations of chromosomes 3 and 8 frequently occur in UM and predict metastatic relapse in patients [6].We determined the genomic risk in patients' tumor samples and compared this with the tumor take rate in mice (Table 2).High risk (HR) of metastases was defined by the loss of chromosome 3 (chr3) and gain of chromosome 8q in patients [6,27].The tumor take was around 50% in high-risk patients whose primary tumors displayed both loss of chr3 and gain of 8q (Table 2).In patients with an intermediate risk (IR, either loss of chr3 or gain of chr 8q in the primary tumor), the tumor take rate was 30%.In four patients, chromosome 3 and 8 status was normal.Only one PDX was obtained from these samples, but it stopped growing at passage 1. In the metastatic patient samples, the take rate was 100% for high-risk tumors, 60% for intermediate-risk, and 0% for low-risk.We thus observed a correlation between tumor take rate and genomic risk as determined by patients' tumors.One PDX belonging to the high-risk group stopped growing at passage 1.We decided to set aside this tumor for further investigations.The status of chromosome 3 and chromosome 8 and the risk of metastatic relapse for patients whose PDXs were analyzed were reported in Table 2.All PDXs obtained correspond to patients with high or intermediate risk of metastases. In conclusion, establishing avatar PDX models for high-risk patients was more successful than for low-risk patients.The time needed to obtain the genetic profile and histology of both the patient's tumor and the corresponding PDX was less than two weeks in our center.This is therefore not a limiting factor for the use of PDX models as avatars for personalized medicine. Next, we evaluated the time required for the samples that were successfully engrafted to reach 60 mm 3 , which is the volume to be reached to start treatments.This interval was 6 to 11 months for PDXs grown from primary tumor samples and 3 to 13 months for those obtained from metastases (Table 3).Genomic risk did not have an impact on this time interval.Our fastest PDX model (MM270) reached a tumor volume of >60 mm 3 within three months.A time interval of 2 weeks to obtain the molecular profile was thus convenient for all PDXs. ",
"section_name": "Genomic Risk of the Patient Correlates with Tumor Take Rate in Mice",
"section_num": "3.3."
},
{
"section_content": "Avatar PDX models can be used to predict response to treatment only if they reliably reflect the patient's tumor.To determine the extent to which the PDX models maintained the features of the corresponding patient tumors, morphologic and molecular alterations characteristic of UM were studied. The histomorphology of the PDXs were compared to the corresponding patient tumors by an expert pathologist.As shown in Supplementary Table S1 and Supplementary Figure S1, the histology of patient tumors showed a profile of either epithelioid cells or a mixed type containing both epithelioid and spindle cells.This profile was conserved in all corresponding PDX models, except in the MP257 model, in which epithelioid cells, the more aggressive ones, became dominant in the PDX. Copy number variation (CNV) studies of tumors are shown in Figure 2 and Supplementary Table S1. Figure 2 shows the whole chromosomes of two PDXs from two primary tumors, MP258 and 271, and two PDXs from metastases, MM252 and MM257.Genomic alterations were conserved between patient tumors and their corresponding PDX models.Notably, the two main chromosomes involved in UM, chromosomes 3 and 8, showed the same alterations between the patients and the PDXs (Supplementary Figure S2 and Supplementary Table S1), except for the MP264 and MP262 models, which showed no gain of chromosome 8q in the patient tumors while the PDXs displayed a gain of 8q.However, in these two models, a subclone with a gain of chromosome 8q was detected in the patient's tumor.Finally, we characterized the genomic alterations that are recurrently found in UM: mutually exclusive mutations in GNAQ or GNA11, as well as mutations in the BRCA1 associated protein-1 (BAP1) and splicing factor 3b subunit 1 (SF3B1).Patient tumors and their corresponding PDXs showed concordance for all GNAQ, GNA11, BAP1, and SF3B1 mutations (Supplementary Table S1). In conclusion, PDX models reliably reproduced the major histological and genomic features of patient tumors and could potentially serve as avatar models to assess the best therapeutic options. ",
"section_name": "Conservation of Histological and Genomic Features between Patient's Tumor and Their PDX",
"section_num": "3.4."
},
{
"section_content": "Next, we wished to assess if it was feasible to use the PDX models to test several drugs, selected based on the molecular alterations of the tumor, before the relapse of the patient, and thus advise the physician on the treatment to use.For this, the first six tumors obtained at passage 0 were grafted into 20 SCID mice each (=passage 1).The time to reach 60 mm 3 , the volume at which we started drug treatment, was between 3 weeks and 13 months for PDXs obtained from metastatic patient tumors, and between 2.5 and 11 months for PDX obtained from primary tumors.This time interval was shorter in passage 1 compared to passage 0. Once this tumor volume had been reached, the treatments were initiated.For this proof-of-concept study, we chose dacarbazin, an alkylating chemotherapeutic agent approved for the first-line treatment of metastatic UM patients, and everolimus, an mTOR inhibitor that is currently being approved for the treatment of certain types of renal, pancreatic, and breast cancers and that had previously showed some efficacy in UM PDXs [28].The mice were treated for 4 weeks; relative tumor volume (RTV) was assessed (Figure 3) and tumor growth inhibition (TGI) was calculated.Among the eleven PDXs obtained, six were treated with both drugs: three PDXs obtained from primary tumors (MP255, MP258, and MP271) and three PDXs obtained from metastases (MM252, MM257, and MM270).The tumor growth rate was highly variable: between 30 and 60% for PDXs from primary tumors and between 50 and 95% for PDXs obtained from patient metastases.The number of mice included in each group varied from two to four mice per group for the ones obtained from primary tumors and from three to seven for those obtained from metastases.Finally, we characterized the genomic alterations that are recurrently found in UM: mutually exclusive mutations in GNAQ or GNA11, as well as mutations in the BRCA1 associated protein-1 (BAP1) and splicing factor 3b subunit 1 (SF3B1).Patient tumors and their corresponding PDXs showed concordance for all GNAQ, GNA11, BAP1, and SF3B1 mutations (Supplementary Table S1). In conclusion, PDX models reliably reproduced the major histological and genomic features of patient tumors and could potentially serve as avatar models to assess the best therapeutic options. ",
"section_name": "Using Avatar Models to Direct Patient Therapy",
"section_num": "3.5."
},
{
"section_content": "Next, we wished to assess if it was feasible to use the PDX models to test several drugs, selected based on the molecular alterations of the tumor, before the relapse of the patient, and thus advise the physician on the treatment to use.For this, the first six tumors obtained at passage 0 were grafted into 20 SCID mice each (=passage 1).The time to reach 60 mm 3 , the volume at which we started drug treatment, was between 3 weeks and 13 months for PDXs obtained from metastatic patient tumors, and between 2.5 and 11 months for PDX obtained from primary tumors.This time interval was shorter in passage 1 compared to passage 0. Once this tumor volume had been reached, the treatments were initiated.For this proof-of-concept study, we chose dacarbazin, an alkylating chemotherapeutic agent approved for the first-line treatment of metastatic UM patients, and everolimus, an mTOR inhibitor that is currently being approved for the treatment of certain types of renal, pancreatic, and breast cancers and that had previously showed some efficacy in UM PDXs [28].The mice were treated for 4 weeks; relative tumor volume (RTV) was assessed (Figure 3) and tumor growth inhibition (TGI) was calculated.Among the eleven PDXs obtained, six were treated with both drugs: three PDXs obtained from primary tumors (MP255, MP258, and MP271) and three PDXs obtained from metastases (MM252, MM257, and MM270).The tumor growth rate was highly variable: between 30 and 60% for PDXs from primary tumors and between 50 and 95% for PDXs obtained from patient metastases.The number of mice included each group varied from two to four mice per group for the ones obtained from primary tumors and from three to seven for those obtained from metastases.Among the three PDXs obtained from patient metastases, MM252 showed a significant response to everolimus with TGI = 55% (p = 0.0043), while no response was observed to dacarbazin.MM270 showed a high antitumor response to both dacarbazin and everolimus with TGIs = 69% and 78%, respectively (Figure 3).But the number of mice in these groups was too small to reach statistical significance.In the last PDX model, MM257, both drugs tended to stabilize tumor growth, but no significant difference was obtained within 4 weeks, due to slow tumor growth. ",
"section_name": "Using Avatar Models to Direct Patient Therapy",
"section_num": "3.5."
},
{
"section_content": "PDX models can be used to choose the best treatment option only if the mice can be treated before the patient relapses.This is a challenge in UM, where relapse often occurs early and prognosis is poor.To define if our PDXs could be used as an avatar model in uveal melanoma, we compared the time required for PDX establishment and treatment with the patients' follow-up data (Figure 4).The follow-ups started at the moment of the surgery (primary tumor or metastases) which allowed for the grafting of the patient's tumor into the mice (=Passage 0).In Figure 4, three key time points are indicated for the PDXs: the first passage (blue), the start of treatment (light green), which corresponds to a tumor volume between 60 and 200 mm 3 , and the end of treatment (yellow), for which the duration was set at 4 weeks.The patients were followed for 3 years after the surgery and three events were specified: tumor relapse, latest news, and/or death.Among the three PDXs obtained from primary human tumors, MP258 showed a mild response to dacarbazin (TGI = 49%) and a significant response to everolimus (TGI = 67%, p = 0.029).The other two PDXs obtained from primary human tumors (MP255 and MP271) grew slowly and 4 weeks of treatments were insufficient to obtain an antitumor effect (Figure 3). Among the three PDXs obtained from patient metastases, MM252 showed a significant response to everolimus with TGI = 55% (p = 0.0043), while no response was observed to dacarbazin.MM270 showed a high antitumor response to both dacarbazin and everolimus with TGIs = 69% and 78%, respectively (Figure 3).But the number of mice in these groups was too small to reach statistical significance.In the last PDX model, MM257, both drugs tended to stabilize tumor growth, but no significant difference was obtained within 4 weeks, due to slow tumor growth. ",
"section_name": "Follow-Up of Patient and Corresponding PDX Models",
"section_num": "3.6."
},
{
"section_content": "PDX models can be used to choose the best treatment option only if the mice can be treated before the patient relapses.This is a challenge in UM, where relapse often occurs early and prognosis is poor.To define if our PDXs could be used as an avatar model in uveal melanoma, we compared the time required for PDX establishment and treatment with the patients' follow-up data (Figure 4).The follow-ups started at the moment of the surgery (primary tumor or metastases) which allowed for the grafting of the patient's tumor into the mice (=Passage 0).In Figure 4, three key time points are indicated for the PDXs: the first passage (blue), the start of treatment (light green), which corresponds to a tumor volume between 60 and 200 mm 3 , and the end of treatment (yellow), for which the duration was set at 4 weeks.The patients were followed for 3 years after the surgery and three events were specified: tumor relapse, latest news, and/or death.The column \"status\" indicates if the PDX avatar could have been used to select a drug for the patient at first relapse (green) or at a subsequent relapse (orange).Red indicates that the patient was deceased before treatment response of PDX was available.Asterisk: PDX models that were treated with dacarbazin and everolimus. In addition to the six PDXs that were treated with DTIC and everolimus, we added five more PDX models to Figure 4.These PDX models were not treated, but the three indicated key time points (the time of first passage, the time to reach the volume of 60-200 mm 3 , and the end of treatment) were extrapolated from their growth curves during the passages.Among these 11 patients, we determined if the response to treatment in the PDX model was available before the patient relapsed and could thus have been useful for the treatment of the patients.This is illustrated in Figure 4 by a color code.Green corresponds to a PDX that could have helped in selecting the best drug for the patient at the first relapse after surgery (end of treatment (in yellow) occurred before relapse (R)); the orange color corresponds to those cases where the PDX response to treatment was not available at the first relapse but could have been used at subsequent relapses (end of treatment in yellow occurred after relapse 1 but before a subsequent relapse); and finally, the red color indicates those cases in which the patient was deceased before the PDX treatment was completed (end of treatment in yellow occurred after death (D)). Among the six PDXs obtained from primary tumors, the treatment response of mice could have been used in five patients: for three patients at their first relapse (metastases appeared) (MP255, MP271, and MP264) and for two patients at their second relapse (MP254 and MP258).Only one model, MP262, could not have been used as an avatar model, since the patient was deceased before treatment could be initiated on mice.The column \"status\" indicates if the PDX avatar could have been used to select a drug for the patient at first relapse (green) or at a subsequent relapse (orange).Red indicates that the patient was deceased before treatment response of PDX was available.Asterisk: PDX models that were treated with dacarbazin and everolimus. In addition to the six PDXs that were treated with DTIC and everolimus, we added five more PDX models to Figure 4.These PDX models were not treated, but the three indicated key time points (the time of first passage, the time to reach the volume of 60-200 mm 3 , and the end of treatment) were extrapolated from their growth curves during the passages.Among these 11 patients, we determined if the response to treatment in the PDX model was available before the patient relapsed and could thus have been useful for the treatment of the patients.This is illustrated in Figure 4 by a color code.Green corresponds to a PDX that could have helped in selecting the best drug for the patient at the first relapse after surgery (end of treatment (in yellow) occurred before relapse (R)); the orange color corresponds to those cases where the PDX response to treatment was not available at the first relapse but could have been used at subsequent relapses (end of treatment in yellow occurred after relapse 1 but before a subsequent relapse); and finally, the red color indicates those cases in which the patient was deceased before the PDX treatment was completed (end of treatment in yellow occurred after death (D)). Among the six PDXs obtained from primary tumors, the treatment response of mice could have been used in five patients: for three patients at their first relapse (metastases appeared) (MP255, MP271, and MP264) and for two patients at their second relapse (MP254 and MP258).Only one model, MP262, could not have been used as an avatar model, since the patient was deceased before treatment could be initiated on mice. Among the five PDXs obtained metastatic patients, only two PDXs, MM267 and MM278, could have been used to select a drug for their corresponding patients, and not at first relapse. ",
"section_name": "Follow-Up of Patient and Corresponding PDX Models",
"section_num": "3.6."
},
{
"section_content": "In this study, we evaluated the feasibility of using PDX models as an avatar to select the best treatment for patients suffering from UM.About half of UM patients develop metastasis, within a time frame that varies from a few months to several years after the diagnosis of the primary disease.Once metastatic, overall survival of UM patients is around 12 months.The timeframe to develop PDX models from metastatic tissue is thus limited, and metastatic tissue is not always available.For this reason, we here evaluated establishing PDX models both from primary tumors and from metastatic tumors. Using twenty-nine patient samples, among which twenty primary UMs and nine liver metastases, we show that tumor take was better for samples obtained from liver metastases than from primary UM, confirming our previous results in UM [29] as well as observations by others in lung, pancreas, prostate, brain, and colon cancer models ( [30,31], reviewed in [32]).This might reflect the fact that metastatic tumors are more frequently high-risk and aggressive tumors, with increased capacity to adapt to a new environment and thus to grow serially in mice.However, once established, we observed that the time for a PDX to grow to a volume of 60 mm 3 and thus to initiate drug treatment was not significantly different between metastases and primary UM. Next, we assessed the histology and molecular alterations of the PDX models as compared to the patient samples.Indeed, in our study, the tumors were grafted into the interscapular fat pad, which does not provide the same microenvironment as the liver.However, it is a well vascularized environment, and it facilitates the follow-up of tumor growth.Orthotopic engraftment into the liver or the eye was not an option here, because it is an invasive procedure requiring expensive imaging and repeated anesthesia, with a high risk of infection or death of the animal and thus loss of the tumor sample.We have previously shown that PDXs grafted into the interscapular fat pad remain very similar to the human tumor in terms of histology and molecular biology [29,31,32].Thanks to the organization that was put in place at Institut Curie for the SHIVA01 trial [25,26], alongside genomics platforms, the time to analyze the histology and the molecular alterations of PDX and patient samples was as short as 2 weeks.In concordance with our previous results, we show a very high similarity between the patient samples and their corresponding PDX models at the level of histology, copy number alterations and genomic variations, both for models derived from primary tumors and from metastases.This confirms our previous observations [29,33] and validates the reliability and clinical value of our UM models.Interestingly, two PDX models showed a gain of 8q that was present as a subclone in the primary patient tumor.This genomic alteration is associated with increased metastatic risk [6].We hypothesize that this was an aggressive subclone of the primary tumor that had a growth advantage upon engraftment in the mice [34,35].Moreover, it might have been the clone leading to metastasis in the patient, as has been observed previously [35][36][37], in which case the PDX model would have been extremely helpful. Finally, we evaluated the feasibility of obtaining clinically relevant data from PDX models within a time frame that is compatible with the evolution of the patients' disease.For this feasibility study, we here decided to treat the mice with two standardized treatments: dacarbazin, an alkylating chemotherapeutic agent that is approved for first-line treatment of metastatic UM, and everolimus, an mTOR inhibitor that had previously showed some efficacy in UM PDX models [28,38].We fixed our treatment duration at 4 weeks.We show that, for the PDXs derived from the primary tumors, drug response could have been available at the first relapse after the surgery for three out of six patients.For two other patients, the chosen treatment could have been used after the second relapse.The remaining patient showed rapid disease evolution and passed away before the PDX could be treated.In the metastatic patients, none of the six PDXs could have been informative at the first relapse, although two out of could have been used at subsequent relapses.In conclusion, PDXs from primary tumors could have been used as avatar models in 50% of the cases if we consider only the first relapse and 85% if we consider all relapses, against 0% and 40%, respectively, for PDXs derived from metastases.We noticed that 4 weeks of treatment was too short in most mice to obtain a frank tumor response, due to the slow growth of the PDX models, and that 6 or 8 weeks of treatment would probably have been preferable.Importantly, in our models, an extension of the treatment length by two or four weeks would not have modified our conclusions in terms of feasibility and utility for the patient for PDXs obtained from primary tumors. In this study, we demonstrate for the first time the technical feasibility of developing a PDX avatar model from UM primary tumor samples, and to test multiple drugs in parallel in order to select the best drug to treat the patient.The clinical interest of PDX models derived from primary tumors has been questioned in the literature because genomic evolution that differed from the one observed in humans has been detected in mice [39].However, UM is a very genetically stable disease [24,40], with few genomic evolutions between the primary tumor and the metastases.This exceptional genetic stability of UM over time, shown by us and others [24,40], allows us to envisage the use of primary tumors to explore treatment options for patients once they become metastatic.A similar avatar approach is currently under clinical evaluation in indications such as breast cancer (NCT05464082), non-smallcell lung cancer (NCT03134456), mantle cell lymphoma (NCT03219047-2018-2023), and pancreatic cancer (NCT04373928), and would be worth testing in UM as well.The only limitation so far is that the mice used in our study are immunodeficient and can therefore not be used to test immunotherapies such as the recently approved tebentafusp [41].However, the development of humanized mouse models may be able to overcome this limitation in the near future [42]. The fraction of patients that could benefit from the avatar approach depends on the tumor take rate and the time required to grow and treat the mouse model.Our results show a take rate of six out of sixteen (38%) among primary UM tumors displaying a high or intermediate genomic risk of relapse.Among these six established models, response to treatment was available before the patient relapsed in five out of six models (83%).These findings should be confirmed in more patients but suggest that the avatar model could be useful in 31% of primary UM patients with high or intermediate risk of relapse.This patient population has the highest need for new personalized treatment options and would be the population of choice to evaluate the UM avatar approach prospectively in the clinical setting.In conclusion, one out of three UM patients with high or intermediate risk of relapse could benefit from avatar models to select in advance the best treatment option to be used at the moment of relapse.This would represent an opportunity in this disease where treatment options are limited. ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/curroncol30100657/s1,Table S1: Comparison of patient samples and PDX models in terms of histology, copy number variation (CNV) and mutation status; ",
"section_name": "Supplementary Materials:",
"section_num": null
},
{
"section_content": "The study was supported \"Carnot Curie Cancer \". Institutional Review Board Statement: Experimental procedures on animals were approved by the ethics committee of the Institut Curie CEEA-IC #118 (Authorization APAFiS #25870-2020060410487032-v1 given by National Authority) in compliance with the international guidelines. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. ",
"section_name": "Funding:",
"section_num": null
}
] |
[
{
"section_content": "The authors thank Ivan Bièche, Aurélie Thuleau, and the animal core facility of the Institut Curie for their contributions.The study was supported by Carnot Curie Cancer. ",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "The data presented in this study are available on request from the corresponding author.The data are not publicly available due to patient privacy and GDPR regulations. ",
"section_name": "Data Availability Statement:",
"section_num": null
},
{
"section_content": "The authors declare no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.2196/42421
|
The Differentially Expressed Genes Responsible for the Development of T Helper 9 Cells From T Helper 2 Cells in Various Disease States: Immuno-Interactomics Study
|
<jats:sec> <jats:title>Background</jats:title> <jats:p>T helper (Th) 9 cells are a novel subset of Th cells that develop independently from Th2 cells and are characterized by the secretion of interleukin (IL)-9. Studies have suggested the involvement of Th9 cells in variable diseases such as allergic and pulmonary diseases (eg, asthma, chronic obstructive airway disease, chronic rhinosinusitis, nasal polyps, and pulmonary hypoplasia), metabolic diseases (eg, acute leukemia, myelocytic leukemia, breast cancer, lung cancer, melanoma, pancreatic cancer), neuropsychiatric disorders (eg, Alzheimer disease), autoimmune diseases (eg, Graves disease, Crohn disease, colitis, psoriasis, systemic lupus erythematosus, systemic scleroderma, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, atopic dermatitis, eczema), and infectious diseases (eg, tuberculosis, hepatitis). However, there is a dearth of information on its involvement in other metabolic, neuropsychiatric, and infectious diseases.</jats:p> </jats:sec> <jats:sec> <jats:title>Objective</jats:title> <jats:p>This study aims to identify significant differentially altered genes in the conversion of Th2 to Th9 cells, and their regulating microRNAs (miRs) from publicly available Gene Expression Omnibus data sets of the mouse model using in silico analysis to unravel various pathogenic pathways involved in disease processes.</jats:p> </jats:sec> <jats:sec> <jats:title>Methods</jats:title> <jats:p>Using differentially expressed genes (DEGs) identified from 2 publicly available data sets (GSE99166 and GSE123501) we performed functional enrichment and network analyses to identify pathways, protein-protein interactions, miR-messenger RNA associations, and disease-gene associations related to significant differentially altered genes implicated in the conversion of Th2 to Th9 cells.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>We extracted 260 common downregulated, 236 common upregulated, and 634 common DEGs from the expression profiles of data sets GSE99166 and GSE123501. Codifferentially expressed ILs, cytokines, receptors, and transcription factors (TFs) were enriched in 7 crucial Kyoto Encyclopedia of Genes and Genomes pathways and Gene Ontology. We constructed the protein-protein interaction network and predicted the top regulatory miRs involved in the Th2 to Th9 differentiation pathways. We also identified various metabolic, allergic and pulmonary, neuropsychiatric, autoimmune, and infectious diseases as well as carcinomas where the differentiation of Th2 to Th9 may play a crucial role.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p>This study identified hitherto unexplored possible associations between Th9 and disease states. Some important ILs, including CCL1 (chemokine [C-C motif] ligand 1), CCL20 (chemokine [C-C motif] ligand 20), IL-13, IL-4, IL-12A, and IL-9; receptors, including IL-12RB1, IL-4RA (interleukin 9 receptor alpha), CD53 (cluster of differentiation 53), CD6 (cluster of differentiation 6), CD5 (cluster of differentiation 5), CD83 (cluster of differentiation 83), CD197 (cluster of differentiation 197), IL-1RL1 (interleukin 1 receptor-like 1), CD101 (cluster of differentiation 101), CD96 (cluster of differentiation 96), CD72 (cluster of differentiation 72), CD7 (cluster of differentiation 7), CD152 (cytotoxic T lymphocyte–associated protein 4), CD38 (cluster of differentiation 38), CX3CR1 (chemokine [C-X3-C motif] receptor 1), CTLA2A (cytotoxic T lymphocyte–associated protein 2 alpha), CTLA28, and CD196 (cluster of differentiation 196); and TFs, including FOXP3 (forkhead box P3), IRF8 (interferon regulatory factor 8), FOXP2 (forkhead box P2), RORA (RAR-related orphan receptor alpha), AHR (aryl-hydrocarbon receptor), MAF (avian musculoaponeurotic fibrosarcoma oncogene homolog), SMAD6 (SMAD family member 6), JUN (Jun proto-oncogene), JAK2 (Janus kinase 2), EP300 (E1A binding protein p300), ATF6 (activating transcription factor 6), BTAF1 (B-TFIID TATA-box binding protein associated factor 1), BAFT (basic leucine zipper transcription factor), NOTCH1 (neurogenic locus notch homolog protein 1), GATA3 (GATA binding protein 3), SATB1 (special AT-rich sequence binding protein 1), BMP7 (bone morphogenetic protein 7), and PPARG (peroxisome proliferator–activated receptor gamma, were able to identify significant differentially altered genes in the conversion of Th2 to Th9 cells. We identified some common miRs that could target the DEGs. The scarcity of studies on the role of Th9 in metabolic diseases highlights the lacunae in this field. Our study provides the rationale for exploring the role of Th9 in various metabolic disorders such as diabetes mellitus, diabetic nephropathy, hypertensive disease, ischemic stroke, steatohepatitis, liver fibrosis, obesity, adenocarcinoma, glioblastoma and glioma, malignant neoplasm of stomach, melanoma, neuroblastoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, and stomach carcinoma.</jats:p> </jats:sec>
|
[
{
"section_content": "CD4 + T helper (Th) cells have been classified into different subsets based on the cytokine profile that each subset secretes and their distinct role in regulating immunity and inflammation.Previous studies have shown that immune cells play a role in various metabolic [1][2][3] and infectious [3][4][5][6][7] diseases.Th9 cells are a subset of CD4 + Th cells that develop from naïve T cells and release interleukin (IL)-9.The generation of Th9 cells from naïve Th0 cells requires a Th2 state as an intermediate.While both Th2 and Th9 cells express PU.1 (spleen focus forming virus [SFFV] proviral integration oncogenes), IRF4 (interferon regulatory factor 4), and GATA3 (GATA binding protein 3), the latter have upregulated expression of IRF4 and suppressed PU.1.The Th2 cells, generated during Th0 cell differentiation, further evolve into Th9 cells in the presence of activated Smad3/Smad4 and IRF4 pathways.The prolonged transforming growth factor beta (TGFβ) stimulation transforms the Th2 cells into Th9 cells and alters the cytokine secretion pattern from an IL-4-dominant phenotype to an IL-9-dominant one [8].Th9 cells produce IL-9, which is crucial in regulating autoimmune and allergic reactions [9].Various other cytokines also affect the development of Th9 cells and IL-9 production.IL-23 inhibits IL-9 production, whereas IL-1 and IL-33 stimulate the production of IL-9 in T cells [10,11].Similarly, IL-25 stimulates the release of IL-9 from T cells [12].In addition, costimulatory receptors, such as OX40, have been found to be a stimulant for the development of Th9 cells [13].Thus, the development of Th9 cells is a result of integrating multiple positive and negative signals in the form of cytokines and costimulation from surface receptors. Th9 cells can manifest differently in various diseases.Th9 cells have been demonstrated to incite allergic airway disease [14].Th9 cells have also been implicated in tumor immunity [10].Interestingly, the evolution of Th2 to Th9 cells does influence the pathophysiology of multiple diseases.The nitric oxide-mediated airway inflammation has been attributed to the inducing effect of nitric oxide on the development of Th9 cells [15].The tricarboxylic acid cycle metabolite succinate stimulates Th9 cell differentiation and leads to Th9 cell-mediated tumor regression.Similarly, Th9 differentiation resulting from IL-35 stimulation accentuates the inflammatory process and leads to an immunoglobulin (Ig) class switch toward IgG4 in IgG4-related diseases [16]. Unfortunately, the experimental approach to Th9 cells has been riddled with difficulty, because a selective deficiency model for Th9 lineage has not yet been defined.In addition, factors needed to develop Th9 cells such as IL-4 and IRF4 are required to develop other Th subsets [17].Our study aimed to compare the transcriptome of Th2 and Th9 cells to identify the pattern of changes in the expression of various genes when the Th2 cells get differentiated into Th9 cells.We also aimed to assess these genes, which are markedly altered in the transition of Th2 to Th9 cells, in various other diseases to enlist the possible diseases in which Th9 cells may play a crucial role. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "We performed a search in the Gene Expression Omnibus (GEO) database using several keywords, including \"Healthy Control,\" \"Wild Type,\" \"Mice,\" \"Mus musculus,\" \"Th9,\" \"Th2,\" and \"Expression profiling by array\" from January 1, 2012, to December 17, 2020, and selected 2 gene series expressions (GSEs) data for further study: GSE99166 and GSE123501.GSE99166 contained 4 samples of Th2 wild-type cells (GSM2634701, GSM2634702, GSM2634711, and GSM2634712) and 5 samples of Th9 wild-type cells (GSM2634695, GSM2634703, GSM2634704, GSM2634713, and GSM2634714) from the spleen.GSE123501 contained 2 samples of Th2 wild-type cells (GSM3505597 and GSM3505602) and another 2 samples of Th9 wild-type cells (GSM350598 and GSM3505603) from the spleen (Figure 1). ",
"section_name": "Expression Profiling: Gene Expression Omnibus Assay to Data Mining for Th2 to Th9 Cells Differentiation",
"section_num": null
},
{
"section_content": "The differentially expressed genes (DEGs) were obtained from the 13 samples of 2 different data sets (GSE99166 and GSE123501) using the GREIN (GEO RNA-seq Experiments Interactive Navigator) platform (BD2K-LINCS Data Coordination and Integration Center).This interactive online web tool analyses GEO RNA-seq data [18].The DEGs extracted from the data sets comprised genes from Th2 and Th9 cells.As we wanted to assess the alteration of genes during the conversion of Th2 to Th9 cells, the analysis was performed with DEGs of Th2 cells as the standard to which DEGs of Th9 cells were compared.The workflow for the data processing and analysis is portrayed in Figure 2. The DEGs were considered upregulated when the expression of genes in Th9 cells was higher than that in Th2 cells.The cutoff for the selection was kept at P<.05, and overlapping DEGs between 2 data sets (GSE99166 and GSE123501) on comparison of Th2 and Th9 cells were identified by the Venn diagram tool [19,20].In addition, the common upregulated, downregulated, and oppositely regulated DEGs of these 2 data sets (GSE99166 and GSE123501) were identified.The fold change expression distribution was visualized by a heat map and violin plot using the Linear Models for the Microarray Data (limma) Package of R (R Foundation for Statistical Computing) and Orange Data Mining (University of Ljubljana) [21,22]. ",
"section_name": "Assortment and Identification of Codifferentially Expressed Messenger RNAs From the Spleen (2 Different) Data Sets",
"section_num": null
},
{
"section_content": "The codifferential genes were divided into 3 parts, namely, (1) common upregulated, (2) common downregulated, and (3) common, oppositely regulated.The top ranked ontological features of all DEGs were analyzed with STRING.The Gene Ontology (GO) terms included the following 3 categories: biological processes, cellular components, and molecular functions.The significant GO terms regulating genes are presented in a radar graph with a negative log10 (false discovery rate).We defined P<.05 as a significant value. ",
"section_name": "Functional Enrichment of Gene Ontology for Common, Regulated DEGs",
"section_num": null
},
{
"section_content": "We searched the functionally significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for top ranked significantly altered DEGs using the STRING and WikiPathways databases.We identified important genes participating in each pathway, and selected the top 7 pathways based on negative log10 (false discovery rate) and P values (<.05) that were important for further study. ",
"section_name": "Kyoto Encyclopedia of Genes and Genomes Pathway Analysis of Top Ranked Significant, Common, Regulated DEGs",
"section_num": null
},
{
"section_content": "We downloaded the complete gene list of the top ranked 7 individual pathways with an interaction network from the KEGG database.We revisualized and constructed the pathway with the help of Cytoscape (Cytoscape Team/Institute for Systems Biology; an open-source software platform for visualizing complex networks and integrating these with any type of attribute data) [23] and marked the DEGs that play a significant role in the differentiation of Th2 to Th9 cells. ",
"section_name": "Genes Assortment and Construction of a Protein-Protein Interaction Network of the Top Enriched Pathways",
"section_num": null
},
{
"section_content": "The top 10 microRNAs (miRs) that targeted the hub genes were predicted by the well-established miR target prediction database miRNet version 22.0 [24], with special emphasis on the selected organism.Default values for the degree of interaction and betweenness were selected.Common miRs and their targeted messenger RNAs (mRNAs) of all groups were sorted by the Venn diagram. ",
"section_name": "Identification of Top Regulatory MicroRNAs Involved in the Th2 to Th9 Differentiation Pathways",
"section_num": null
},
{
"section_content": "The DEGs that were identified to play a significant role in Th2 to Th9 differentiation were further analyzed for their involvement in various pathways pertaining to specific diseases using DisGeNET (IBI Group) [25], a discovery platform that describes genes, transcription factors (TFs), chemokines, and IL in association with various specific diseases. ",
"section_name": "Construction of a Gene-Disease-Based Genomic Pathway Interaction Network",
"section_num": null
},
{
"section_content": "The study was approved by the Institutional Ethics Committee of All India Institute of Medical Sciences (AIIMS) Jodhpur (certificate reference number AIIMS/IEC/2019-20/792). ",
"section_name": "Ethical Considerations",
"section_num": null
},
{
"section_content": "",
"section_name": "XSL • FO",
"section_num": null
},
{
"section_content": "",
"section_name": "RenderX",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "The Mus musculus (C57BL/6) mRNA expression profiles of GSE99167 and GSE123501, which were selected for this study, included the expression profiles of Th2 and Th9 cells obtained from the spleen.We extracted and compared mice spleen samples from 2 different studies to identify genes that are involved in the differentiation of Th2 to Th9 cells.In both groups, 254 common mRNAs were identified, and 634 common DEGs were identified, of which 236 were downregulated and 260 were upregulated.We performed a quality assessment of the selected samples for our expression profiles (Figures 3A-3I; see Tables S1 andS2 in Multimedia Appendix 1, and Multimedia Appendix 2 for larger version of figures).(cluster of differentiation 197), IL-1RL1 (interleukin 1 receptor-like 1), CD101 (cluster of differentiation 101), CD96 (cluster of differentiation 96), CD72 (cluster of differentiation 72), CD7 (cluster of differentiation 7), CD152 (cytotoxic T lymphocyte-associated protein 4), CD38 (cluster of differentiation 38), CX3CR1 (chemokine [C-X3-C motif] receptor 1), CTLA2A (cytotoxic T lymphocyte-associated protein 2 alpha), CTLA28, and CD196 (cluster of differentiation 196).In addition, the differential expression of various TFs such as FOXP3 (forkhead box P3), IRF8 (interferon regulatory factor 8), FOXP2 (forkhead box P2), RORA (RAR-related orphan receptor alpha), AHR (aryl-hydrocarbon receptor), MAF (avian musculoaponeurotic fibrosarcoma oncogene homolog), SMAD6 (SMAD family member 6), JUN (Jun proto-oncogene), JAK2 (Janus kinase 2), EP300 (E1A binding protein p300), ATF6 (activating transcription factor 6), BTAF1 (B-TFIID TATA-box binding protein associated factor 1), BAFT (basic leucine zipper transcription factor), NOTCH1 (neurogenic locus notch homolog protein 1), GATA3, SATB1 (special AT-rich sequence binding protein 1), BMP7 (bone morphogenetic protein 7), and PPARG (peroxisome proliferator-activated receptor gamma) may influence the differentiation of Th2 to Th9 cells.The expression of the aforementioned immune regulators is represented by a heat map and Venn diagram in Figures 4A-4F (also see Tables S3-S5 in Multimedia Appendix 1, and Multimedia Appendix 2 for larger version of figures). ",
"section_name": "Assortment of Significant DEGs in the Differentiation of Th2 to Th9 Cells",
"section_num": null
},
{
"section_content": "We downloaded the complete protein-protein interaction (PPI) network of the identified KEGG pathways from the KEGG database.The Cytoscape software was used for the construction of the network.The significantly altered DEGs of cytokines, chemokines, receptors, and TFs were highlighted in the respective networks.Our analysis of the KEGG pathway enrichment and PPI network demonstrated that the genes that had a significantly altered expression in Th9 cells when compared with Th2 cells also played a significant role in other immune regulating pathways.These affected pathways were mainly involved in cytokines-cytokines interaction, Th1 and Th2 differentiation, CTLA4 (cytotoxic T lymphocyte-associated protein 4) regulation, T-cell receptor signaling, Fc epsilon signaling, Th17 cell differentiation, IBD, and cancer.The concurrent presence of these genes in the aforementioned pathways highlights the significance of the differentiation of Th2 to Th9 in diseases where these pathways are affected.The role of the identified DEGs in these pathways and their interaction with other genes has been depicted in Figures 567.See Multimedia Appendix 2 for larger images. ",
"section_name": "Construction of the Protein-Protein Interaction Network of DEGs Involved in the Transition of Th2 to Th9 Cells",
"section_num": null
},
{
"section_content": "We performed a gene similarity analysis to find similar genes in all the 7 KEGG pathways identified with the help of the Venn diagram and calculate the percentage of similarity among the genes that were altered.We observed that 7/13 (54%) genes were similar between the \"Th1 and Th2 cell differentiation\" and \"IBD\" pathways, whereas 7/14 (50%) genes were similar between the \"Th1 and Th2 cell differentiation\" and \"Th17 cell differentiation\" pathways (Figure 7A; see Multimedia Appendix 2 for larger images). ",
"section_name": "Assessment of Gene Similarity in Pathways Identified in the KEGG Pathway Enrichment Analysis",
"section_num": null
},
{
"section_content": "To explore the posttranscriptional regulation of the identified DEGs, we predicted the miRs that could target the identified DEGs.We identified the following 53 common miRs that could target the DEGs listed in our analysis: let-7b-5p, let-7f-5p, let-7g-5p, let-7i-5p, miR-1-3p, miR-101-3p, miR-103a-3p, miR-107, miR-10a-5p, miR-10b-5p, miR-122-5p, miR-124-3p, miR-129-3p, miR-130a-3p, miR-133a-3p, miR-139-5p, miR-147a, miR-155-5p, miR-16-5p, miR-17-5p, miR-186-5p, miR-195-5p, miR-200b-3p, miR-20a-3p, miR-20a-5p, miR-20b-5p, miR-21-3p, miR-21-5p, miR-22-3p, miR-224-5p, miR-24-3p, miR-26a-5p, miR-26b-5p, miR-27-5p, miR-27a-3p, miR-302a, miR-30a-5p, miR-30c-5p, miR-30d-5p, miR-320a, miR-34-5p, miR-374-5p, miR-426, miR-429, miR-618, miR-6499-3p, miR-93-5p, miR-98-5p, miR-103a-3p, miR-139-5p, miR-147a, miR-195-5p, and miR-27a-5p. ",
"section_name": "Prediction of miRs That Target the DEGs Involved in the Transition of Th2 to Th9 cells",
"section_num": null
},
{
"section_content": "Subsequent to the identification of pathways affected as a result of the alteration of DEGs found in our analysis, we further searched for possible diseases whose pathogenesis is affected by alterations in these pathways.We listed the diseases where alterations in the aforementioned 7 pathways have already been documented, and these were as follows: metabolic diseases (eg, diabetes mellitus, diabetic nephropathy, hyperactive behavior, hypertensive disease, ischemic stroke, steatohepatitis, liver fibrosis, obesity), allergic and pulmonary diseases (eg, asthma, chronic obstructive airway disease, chronic rhinosinusitis, nasal polyps, pulmonary hypoplasia, hay fever), carcinomas (eg, acute leukemia and myelocytic leukemia, B-cell lymphomas, lymphoma, adenocarcinoma, breast carcinoma, carcinoma of the lung, cervical cancer, colorectal carcinoma, glioblastoma and glioma, liver carcinoma, malignant neoplasm of the stomach, melanoma, neuroblastoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, stomach carcinoma), neuropsychiatric disorders (eg, mental depression, schizophrenia, Alzheimer disease), autoimmune diseases (eg, Graves disease, Crohn disease, colitis, psoriasis, systemic lupus erythematosus [SLE], systemic scleroderma, rheumatoid arthritis, multiple sclerosis [MS], IBD, atopic dermatitis, eczema), and infectious diseases (eg, sepsis, septicemia, tuberculosis, hepatitis, herpes simplex infections, malaria; Figure 8). ",
"section_name": "Identification of Diseases Associated With Dysregulation of the Identified miRs and DEGs",
"section_num": null
},
{
"section_content": "",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "In this study, we compared 2 different data sets (GSE99166 and GSE123501) that have compared the mRNA expression in Th2 and Th9 cells.We identified common DEGs that have significantly altered expression between Th2 and Th9 cells from these 2 data sets.Sequential assessment of the DEGs and miRs that had significantly altered expression between Th2 and Th9 cells allows to identify disease states that affect the differentiation process.Although this analysis does not answer whether differentiation of Th2 to Th9 is the cause or the effect of the disease state, it does unravel the possibility of hitherto unknown associations between various diseases and the process of differentiation of Th2 to Th9 cells.Our analysis indicates that differentiation of Th2 to Th9 may play a crucial role via the alteration of DEGs (Table 1) and miRs (Table 2) in various metabolic diseases, allergic and pulmonary diseases, carcinomas, neuropsychiatric disorders, autoimmune diseases, and infectious diseases.In concordance with the existing literature, it was revealed that Th9 cells might play a major role in erythematosus, MS, IBDs, and psoriasis.The role of Th9 cells in autoimmune disease has already been explored in multiple studies [26], including in Graves disease [27], Crohn disease [28][29][30], psoriasis [31], SLE [32][33][34][35], systemic scleroderma [36], rheumatoid arthritis [37][38][39][40], MS [26,36,41,42], IBD [26,29,30,43], and atopic dermatitis/eczema [44], which have demonstrated an increased level of differentiation of Th2 to Th9 cells.Th9 cells and IL-9 have been observed in peripheral blood mononuclear cells and synovial fluid from patients with rheumatoid arthritis.Toll-like receptor 2 (TLR2) stimulates naïve CD4 + T cells for IL-9 secretion and Th9 differentiation by increasing the expression of TFs BATF and PU.1.TLR2 activation results in increased expression of IL-33 and its receptor ST2, augmenting IL-9 gene expression and Th9 cell development [45].Similarly, in patients with SLE, Th9 cell differentiation is suppressed by repression of IRF4 expression [46].Although the role of Th9 has been explored in experimental models of MS and IBD, there is insufficient evidence regarding its role in humans.Th9 cells have been shown to play a pathogenic role in experimental autoimmune encephalomyelitis, an animal model of MS [47].However, only limited studies have assessed Th9 cells in human patients with MS.The skin toxicity of Th9 cells makes them a crucial link in the pathophysiology of multiple skin diseases [48].Our study highlights the possibility of Th9 playing a crucial role in the pathophysiology of various autoimmune skin diseases such as eczema, atopic dermatitis, psoriasis, and dermatitis.A predominant expression of IL-9 from Th9 cells was observed to be a characteristic immunologic signature in psoriatic arthritis [49].Similarly, IL-9 and PU.1 gene expressions in atopic dermatitis were higher and associated with disease severity [50].In addition, the Th9 cell percentage in patients with atopic dermatitis correlated with serum IgE levels, highlighting the link between allergy and the development of Th9 cells [51].Our in silico analysis further reiterated the involvement of Th9 in various autoimmune pathways.The involvement of IL-9 and Th9 cells in allergic response can also be seen in other diseases.One such allergic disease in which Th9 cells have been recently explored is asthma.Patients with allergic asthma have increased peripheral blood Th9 cells and elevated levels of serum IL-9 [51].SGK1 (serum/glucocorticoid regulated kinase 1) has been shown to enhance the differentiation of Th9 by modulating the nuclear factor kappa B (NF-κB) signaling pathway in patients with asthma [52].The activation of MAPK (mitogen-activated protein kinase) has also been attributed to the activation of Th9 cells in mice models of asthma [53].Interestingly, IL-9 and IL-13 have been elevated in patients with chronic obstructive airway disease compared with asthma [54].However, so far, the Th9 cells have not been explored for their significance in the pathophysiology of chronic obstructive pulmonary disease.Interestingly, apart from asthma, our in silico analysis highlighted chronic obstructive airway disease, tuberculosis, and chronic rhinosinusitis with nasal polyps as major airway diseases in which Th9 cells may play a crucial role.Our findings are in sync with the study of Ye et al [55], which demonstrated tuberculous pleural effusion to be chemotactic for Th9 cells, while pleural mesothelial cells in tuberculosis stimulated the Th9 cell differentiation.This in silico analysis also highlights the possible role of Th9 in neuropsychiatric diseases.Very few studies have explored the role of Th9 in neuropsychiatric disorders.Saresella et al [56] have demonstrated an increase in the activity of Th9 lymphocytes, while postthymic maturation pathways showed an accumulation of differentiated effector T lymphocytes (CD4 + ).In Alzheimer disease, schizophrenia, and multiple-episode schizophrenia, although IL-9 has been elevated, limited studies have been performed to assess the role of Th9 cells in the pathophysiology of the diseases [56,57].In addition to the aforementioned diseases, this study identified malignancies as one of the disease states that could be affected by the development of Th9 cells.The role of Th9 cells in modulating immunity in cancer has been widely explored.Th9 cells contribute to antitumor immunity by enhancing the recruitment and activation of mast cells, natural killer cells, CD8 T cells, and dendritic cells in the tumor microenvironment.The antitumor effect of Th9 cells has been documented in various animal studies.Lu et al [58] have demonstrated the protective effects of IL-9 and Th9 on tumor development.The tumor-specific Th9 cells promoted the activation of CD8 + cytotoxic T lymphocytes by recruiting dendritic cells into tumor tissues and subsequently presenting tumor antigens in tumor-draining LNs.Th9 cells in tumor tissues mount an inflammatory response via CTL in a CCL20/CCR6 (chemokine [C-C motif] receptor 6)-dependent manner [59,60].Wang et al [61] also demonstrated that Th9-enriched CD4 + T cells significantly increased the expansion of activated CD8 + T cells in a manner that was dependent on the expression of IL-9R (interleukin 9 receptor).Th9 thus seems to enhance antitumor immune response through T-cell cytotoxicity and play a crucial role in controlling the progression of cancer [62].Apart from Th9 cells, the cytokine IL-9 has also been widely explored in cancers.Expression of IL-9 in the serum and circulating CD4 + T cells was significantly upregulated in patients with breast cancer compared with healthy controls [63].Purwar et al [10] demonstrated that IL-9 depletion in RORγt-deficient mice promoted melanoma growth.Zheng et al [64] demonstrated that Th9 cells produce IL-9 to induce glioma cell apoptosis and inhibit tumor growth.Interestingly, tumor-specific Th9 cells displayed a unique PU.1-TRAF6-NF-kB activation-driven hyperproliferative feature, suggesting a persistence mechanism rather than an antiapoptotic strategy.This equips tumor-specific Th9 cells to become a more effective CD4 + T-cell subset for adoptive cancer therapy [65].Although Th9 cells play an important role in tumor suppression, they have not been studied in various cancer subtypes.Our analysis suggests a possible role for Th9 in different cancer types such as malignant neoplasm of the stomach, melanoma, neuroblastoma, osteosarcoma, pancreatic carcinoma, and prostate carcinoma.Finally, our study also highlights the possible role of Th9 in different metabolic diseases.Interestingly, to our knowledge, no study has yet explored the role of Th9 in metabolic diseases such as diabetes and obesity.We want to highlight these lacunae to open up newer research attempts that would explore the role of Th9 in metabolic diseases.The insights into the role of Th9 in metabolic diseases would better help delineate the role of immunological dysregulation in developing metabolic diseases.m TRAF6: TNF receptor-associated factor 6. n IFNAR: interferon (alpha and beta) receptor. o TGF: transforming growth factor. p SMAD: SMAD family member.q PU.1: spleen focus forming virus (SFFV) proviral integration oncogene.r TSLP: thymic stromal lymphopoietin.s TSLPR: thymic stromal lymphopoietin receptor. ",
"section_name": "Principal Findings",
"section_num": null
},
{
"section_content": "The main limitation of the study is that the analysis is based on an in silico method where only a few specific wild-type samples from data sets of previous studies were included; therefore, further validation of the identified genes and miRNAs is required in various animal models and human diseases.The data sets were compiled using different arrays on the Affymetrix platform, which may account for some of the variability in the results.However, the functional enrichment for the mRNAs highlighted some significant pathways related to immune regulation and its derangements. ",
"section_name": "Limitations",
"section_num": null
},
{
"section_content": "This study identified common DEGs of ILs, receptors, and TFs that have significantly altered expression between Th2 and Th9 cells.The KEGG pathway enrichment analysis identified cytokines-cytokines interaction, Th1 and Th2 differentiation, T-cell receptor signaling regulation via CTLA4, Fc epsilon signaling, and Th17 cell differentiation as the significant pathways affected by the identified DEGs.Our study identified hitherto unexplored possible associations between Th9 and disease states.The interactome analysis also identified pathways that are involved in various metabolic diseases, allergic and pulmonary diseases, carcinomas, neuropsychiatric disorders, autoimmune diseases, and infectious diseases, where differentiation of Th2 to Th9 may play a crucial role.The scarcity of studies on the role of Th9 in metabolic diseases highlights the lacunae in this field.Thus, our study provides the rationale for exploring the role of Th9 in various metabolic disorders. ",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "The authors are grateful to the All India Institute of Medical Sciences Jodhpur for providing the research facility to perform this in silico experiment.MK is supported by a senior research fellowship of The University Grants Commission of India (number NOV2017-361200). ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "Publicly available GEO data sets were used for the analysis in this study.These data sets can be accessed online [102,103]. ",
"section_name": "Data Availability",
"section_num": null
},
{
"section_content": "None declared. Fold change expression of the significant differentially expressed genes analyzed. [DOCX File , 64 KB-Multimedia Appendix 1] Higher resolution images for Figures 34567 ©Manoj Khokhar, Purvi Purohit, Ashita Gadwal, Sojit Tomo, Nitin Kumar Bajpai, Ravindra Shukla.Originally published in JMIR Bioinformatics and Biotechnology (https://bioinform.jmir.org),23.02.2023.This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Bioinformatics and Biotechnology, is properly cited.The complete bibliographic information, a link to the original publication on https://bioinform.jmir.org/,as well as this copyright and license information must be included. ",
"section_name": "Conflicts of Interest",
"section_num": null
},
{
"section_content": "None declared. ",
"section_name": "Conflicts of Interest",
"section_num": null
},
{
"section_content": "Fold change expression of the significant differentially expressed genes analyzed. [DOCX File , 64 KB-Multimedia Appendix 1] ",
"section_name": "Multimedia Appendix 1",
"section_num": null
},
{
"section_content": "Higher resolution images for Figures 34567 ©Manoj Khokhar, Purvi Purohit, Ashita Gadwal, Sojit Tomo, Nitin Kumar Bajpai, Ravindra Shukla.Originally published in JMIR Bioinformatics and Biotechnology (https://bioinform.jmir.org),23.02.2023.This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Bioinformatics and Biotechnology, is properly cited.The complete bibliographic information, a link to the original publication on https://bioinform.jmir.org/,as well as this copyright and license information must be included. ",
"section_name": "Multimedia Appendix 2",
"section_num": null
}
] |
10.7892/boris.126363
|
Comprehensive Analysis of Alternative Splicing Across Tumors from 8,705 Patients
|
Our comprehensive analysis of alternative splicing across 32 The Cancer Genome Atlas cancer types from 8,705 patients detects alternative splicing events and tumor variants by reanalyzing RNA and whole-exome sequencing data. Tumors have up to 30% more alternative splicing events than normal samples. Association analysis of somatic variants with alternative splicing events confirmed known trans associations with variants in SF3B1 and U2AF1 and identified additional trans-acting variants (e.g., TADA1, PPP2R1A). Many tumors have thousands of alternative splicing events not detectable in normal samples; on average, we identified ≈930 exon-exon junctions ("neojunctions") in tumors not typically found in GTEx normals. From Clinical Proteomic Tumor Analysis Consortium data available for breast and ovarian tumor samples, we confirmed ≈1.7 neojunction- and ≈0.6 single nucleotide variant-derived peptides per tumor sample that are also predicted major histocompatibility complex-I binders ("putative neoantigens").
|
[
{
"section_content": "und für die Berichterstattung an die Fakultäten der Universität und den Kanton Bern verwendet.(if necessary after consultation with the submitters, cf.below, 6.4).Members of the Editorial Board are appointed by the Open Science Team of the University Library of Bern. • Roles of researchers: Submitters can pick the roles of researchers whose work they submit to BORIS Portal.A role is a combination of name, institute/department, and duration of employment.The role of a researcher is necessary to unambiguously identify contributors to research outputs.• Eligibility: authorization on a technical level to perform certain actions in BORIS Portal (e.g., submit or review items).The eligibility criteria are determined by the Open Science Team of the University Library of Bern.On a technical level, the system administrator can authorizes users (granting of rights).• Open -The datasets are accessible without restrictions and can be downloaded. • Embargo -The records are freely accessible after the expiration of an embargo period.The embargo end date is determined by the submitter. • Restricted -Access to records must be requested from a contact person. • Closed -Records cannot be uploaded to the repository but metadata can be entered. • Metadata Only -If data are already uploaded to another repository, the metadata on BORIS Portal can be used to verify the existence of the dataset. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Reasons to restrict access to records may include legal or ethical constraints, contractual obligation, or other compelling reasons. ",
"section_name": "Access restriction for datasets",
"section_num": "5.2."
},
{
"section_content": "Interested persons may request access to datasets that are under embargo, restricted or closed access.Such requests will be forwarded to the contact person that was designated when the dataset was submitted.The contact person can then grant or deny access.Submitters can specify terms of access by means of a Data Transfer Agreement (DTA). ",
"section_name": "Access to records on request",
"section_num": "5.3."
},
{
"section_content": "• BORIS Portal offers standard licenses for datasets.Depending on the content type, submitters can add other licenses as needed.• Should the submitter choose \"restricted\" or \"closed\" access, there is no dataset license to select.In this case, a Data Transfer Agreement (DTA) can regulate access.• Supplementary materials and Data Transfer Agreements in BORIS Portal are public domain (CC0), publicly accessible and downloadable.5.5.If not stated otherwise, content from BORIS Portal should be cited appropriately for subsequent use and reuse, at least by indicating author, title, date, repository (BORIS Portal), DOI. 6. Submission 6.1.Only designated members (including scientific staff) of the University of Bern or their authorized representatives can submit documents and metadata to BORIS Portal.6.2.The quality, validity, and authenticity of BORIS Portal content are the sole responsibility of the submitter.6.3.The compliance of content shared on BORIS Portal with legal, ethical, and similar regulations and requirements is the sole responsibility of the submitter.6.4.BORIS Portal editorial staff check submissions only for the authorization of the author/submitter, valid layout and format, and ensures the exclusion of content that is not relevant to the scope of BORIS Portal or that is inappropriate (spam).6.5.After review (and if necessary after consultation with the submitters), the BORIS portal editorial team publishes the entered metadata and makes the content accessible according to the access type chosen by the submitter. 7. Storage and availability 7.1.Metadata will be retained for the operational life of BORIS Portal.Should BORIS Portal cease operation, all metadata and content will be transferred to another repository.7.2.Submitted items will be retained for a minimum of 10 years.7.3.File formats may be converted if necessary to preserve the readability of files.7.4.It may not be possible to preserve the readability of files.BORIS Portal reserves the right to work with external partners to convert or migrate file formats, to use software emulations for old file formats, and/or to store metadata for preservation purposes.7.5.The content of the repository is regularly backed up by the university's IT services according to current best practice. 8. Withdrawal of published items 8.1.Published items may be withdrawn only for compelling reasons.Reasons for withdrawal are: • Proven copyright infringement or plagiarism; • Legal provisions or their violation; • National security; • Falsified research. If BORIS Portal receives proof that one of these reasons applies, the corresponding item will be removed as soon as possible.BORIS Portal does not assume any responsibility for breaches of law in the aforementioned sense (see section 3.2) by uploaded content.8.2.Withdrawn items may not be deleted, but will be withdrawn from public visibility.They may be retained for verification purposes.8.3.Withdrawn items may be deleted completely in case of legal violations.8.4.Digital Object Identifiers (DOIs) of withdrawn items will continue to be retained for verification purposes.BORIS Portal can then indicate the status of the relevant submissions as \"deleted\" in harvesting requests.8.5.DOIs continue to be forwarded to so-called \"tombstone\" references that display the metadata of the deleted item.This is done to prevent broken links and to retain item histories.8.6.The metadata of withdrawn items will not be searchable or findable.8.7.Lists of errors (errata) and corrections (corrigenda) can be added to the original entry as needed.9. Data of members of the University of Bern, data protection, and confidentiality 9.1.BORIS Portal processes data about members of the University of Bern.Processing data means in particular storing, linking, disclosing, and deleting data, as necessary for the purposes mentioned in section 2 above.9.2.Eligible employees of the university administration have direct access to BORIS Portal.For eligibility, see above, section 0 (definitions).9.3.All those involved in the submission or review of metadata and data receive instructions on the confidential and secure handling of the entered information.9.4.The following information will be checked against data from the personnel administration system of the University of Bern for the purpose of identification: 1. Name The data under 1-4 can be displayed or viewed publicly.The data under 5 are only visible during submission as well as during review by authorized members of the university administration (see above, 9.2).The data under 6 are treated as confidential and can only be viewed by authorized members of the university administration.9.5.BORIS Portal will also process other data entered.These include: • Externally provided identifiers such as ORCiD numbers; • Information on research projects such as participating researchers and organizational units; • Research project funding information (data submitter can remove this from public visibility); • Documents that can be uploaded for internal purposes (e.g.project management) of the researchers.These documents will be visible only to the owners as well as to the system administrator, unless the owners grant permission to other persons. ",
"section_name": "Licenses",
"section_num": "5.4."
},
{
"section_content": "In accordance with legal provisions, BORIS Portal attaches great importance to the accuracy and timeliness of the stored data.However, BORIS Portal does not guarantee that the stored information is correct, up-to-date, complete, and of adequate quality.BORIS Portal reserves the right to correct or delete information.BORIS Portal shall not be liable for any loss or damage of any kind incurred as a result of accessing, using, or not using the published information.BORIS Portal has not reviewed third party websites, i.e. those that are not on the servers of BORIS Portal or within its sphere of influence, and that are hyperlinked to this website, and assumes no responsibility for their content. 11. Review of these guidelines These guidelines will be reviewed at least annually.Date of last review: August 18, 2021. ",
"section_name": "Disclaimer of warranty and liability",
"section_num": "10."
}
] |
[] |
10.7150/jca.15274
|
Notch Signaling Activation in Cervical Cancer Cells Induces Cell Growth Arrest with the Involvement of the Nuclear Receptor NR4A2
|
Cervical cancer is a second leading cancer death in women world-wide, with most cases in less developed countries. Notch signaling is highly conserved with its involvement in many cancers. In the present study, we established stable cervical cell lines with Notch activation and inactivation and found that Notch activation played a suppressive role in cervical cancer cells. Meanwhile, the transient overexpression of the active intracellular domain of all four Notch receptors (ICN1, 2, 3, and 4) also induced the suppression of cervical cancer Hela cell growth. ICN1 also induced cell cycle arrest at phase G1. Notch1 signaling activation affected the expression of serial genes, especially the genes associated with cAMP signaling, with an increase of genes like THBS1, VCL, p63, c-Myc and SCG2, a decrease of genes like NR4A2, PCK2 and BCL-2. Particularly, The nuclear receptor NR4A2 was observed to induce cell proliferation via MTT assay and reduce cell apoptosis via FACS assay. Furthermore, NR4A2's activation could reverse ICN1-induced suppression of cell growth while erasing ICN1-induced increase of tumor suppressor p63. These findings support that Notch signaling mediates cervical cancer cell growth suppression with the involvement of nuclear receptor NR4A2. Notably, Notch/NR4A2/p63 signaling cascade possibly is a new signling pathway undisclosed.
|
[
{
"section_content": "As the second leading cause of cancer death, cervical cancer is a major health concern for women world-wide (1,2), with most cases occurring in less developed countries.The highest incidences are happening in Latin America, the Caribbean and Africa (World Cancer Research: www.wcrf.org).Cervical cancer is known to be involved with multiple signaling pathways (3)(4)(5)(6).The human papillomavirus (HPV) has been shown to be an essential component in cervical cancer progression (1)(2)(3)(4)7).However, many other factors, such as Notch, Wnt, COX2, NF-KB, p53 and RhoC, are also critical elements associated with the development of cervical cancer (4,(6)(7)(8)(9)(10)(11)(12).Notch signaling especially has been found to play a critical role in cervical cancer development. Notch signaling is highly conserved and is critical for human development.Importantly, Notch signaling is found aberrantly expressed in many types of cancers and is involved in cancer progression.The up-regulation of Notch1 and the cognate ligand, ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "Jagged1, have been demonstrated in cervical cancer cells (2,(13)(14)(15).This prompted an investigation into the possible role of Notch signaling in this cancer progression.Notch signaling activation in cervical cancer further displays suppression on cell proliferation and tumor growth (7,14,16,17).The strategy of targeting Notch signaling provides a potential and effective therapeutic alternative in the treatment of cervical cancer (18)(19)(20).However, Notch signaling is much more complicated.Notch signaling acts differently in different stages of cervical cancer development, with an up-regulation of this signaling in early stages and a down-regulation in its late stage.The controversial effects of Notch signaling, in the same cervical cancer cell models, also are reported by other independent investigators (5,21).Thus, the role of Notch signaling and its precise molecular mechanisms are not completely known. Notch1 activation was also observed to stimulate the signaling of G protein-coupled somatostatin receptors xxx.This characteristic has been applied in combination therapy by combining a Notch signaling activator with a SSTR2-targeting peptide-drug and using them together as a conjugate.The combination therapy displays enhanced anti-tumor activity when compared to each used alone (19,20,22).However, the Notch-mediated mechanism is not yet clear.Also, Notch activation could enhance for skolin-induced cAMP production (19).We hypothesized that genes associated with cAMP signaling may be involved in Notch-mediated signaling networks.In the present study, we further investigated and determined the effects of the active forms of all four Notch receptors on cervical cancer cells.Particularly, we evaluated the effects of Notch1 activation on certain genes associated with cAMP/Ca 2+ signaling via PCR array.We found that the nuclear receptor NR4A2 was down-regulated by Notch activation.NR4A2 activation increased cervical cancer cell growth via acting in an oncogenic role.Targeting crosstalk events of Notch and NR4A2 is likely to provide valuable paradigms around which to develop highly specific chemotherapeutic interventions. ",
"section_name": "Ivyspring International Publisher",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "The plasmids expressing the intracellular domains of the four Notch receptors (ICN, ICN2, ICN3, and ICN4) and dnMAML (DNL) were gifts from Dr. Wu (University of Florida).The plasmids with the genes NR4A2 (Nurr1), Vinculin (VCL), THBS1 (TSP1), p63 and Twist were obtained from Addgene (www.addgene.org),including pCCL-NR4A2 (plasmid 35000) and the controls pCCL (10881), pmEN1-VCL (54304), pmEN1 (53976), pBabe-Twist1 (1783), pBabe (10668), pcDNA-THBS1 (12405) and pcDNA-p63 (27008).Antibodies of p21 (Cat.No.: sc-756), p63 (sc-8343), c-Myc (sc-788), NR4A2 (sc-990), HES1 (sc-25392) and β-actin (sc-1616-HRP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). ",
"section_name": "Materials",
"section_num": null
},
{
"section_content": "Human cervical cancer Hela cells were purchased from ATCC (American Type Culture Collection, Manassas, VA).The stable cancer cell lines Hela-ICN1 (over-expressing ICN1, Notch1 activation) and Hela-DNL1 (over-expressing dominant-negative mastermind-like1 (DNL1, or dnMAML1), Notch inactivation) along with Hela-GFP cells were established as described previously (19).These cells were maintained in MEM medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 0.5% kanamycin.For transient transfection, 0.5ml of a Hela cell suspension with 2x10 5 cells/ml was plated in each well of 24-well plates and cultured overnight.Two µl of Lipofectamine TM 2000 (Lipo-2000) and a total amount (0.4µg) of DNA were added separately into each vial with 50 µl Opti-MEM transfection medium, and combined together after a 5-10 minute incubation.The DNA-Lipo-2000 complexes were mixed well and incubated for 20-30 minutes and then added to each well.Growth medium was replaced 4-5 hours later and cells were incubated for 72-hours. ",
"section_name": "Cell culture and cell transfection",
"section_num": null
},
{
"section_content": "Total RNA was isolated from tumor cells and RT-PCR was performed as described in the protocols (Invitrogen, Carlsbad, CA).The primers and conditions for RT-PCR analyses were as described previously (19).Real-time PCR assays and PCR arrays (cAMP/Ca 2+ signaling pathway (PAHS-066), GPCR signaling pathway (PAHS-071)) (SABiosciences) were performed as described (19).β-actin was used as the internal control and data were analyzed by applying the 2 -∆∆ CT methods. ",
"section_name": "Real-time PCR and PCR array",
"section_num": null
},
{
"section_content": "This assay was employed as described in the protocol (Santa Cruz).Briefly, cells were harvested, re-suspended in RIPA buffer with cocktail inhibitors, homogenized with a 21-gauge needle, mixed with the loading buffer and heated for 7 min at 95 o C. Supernatants were loaded to run on a 8-16% Tris-glycine gel after centrifugation at 13,000xg.Protein was transferred to a nitrocellulose membrane and blocked with 5% fat-free milk, washed and incubated with primary antibody.The membrane was washed again and incubated with second antibody (Santa Cruz).Films were developed according to the ECL system protocol (Amersham Biosciences, England).The experiments were separately done three times. ",
"section_name": "Western blot analysis",
"section_num": null
},
{
"section_content": "The cell proliferation assay (Promega, Madison, WI) was performed as described (19).Briefly, 50 µl aliquots of medium with different concentrations of compounds were added to 96-well plates.All compound concentrations were tested in triplicate.Another 50 µl of the BON cell stock (1x10 5 cells/ml of media) was dispensed into each test well and the plates were incubated at 37 o C in a CO 2 incubator for 3 days.Following the incubation period, 15 µl of the dye solution was added to each well and the plates were then incubated at 37 o C for 4 hours, followed by the addition of 100 µl per well of the solubilization solution.The plates were incubated at 37 o C until the contents in each well became a uniform-colored solution.The absorbance was measured and recorded at 570 nm by a Victor Plate Reader (PerkinElmer, Boston, MA). ",
"section_name": "Cell proliferation",
"section_num": null
},
{
"section_content": "Cells were plated in 6-well plates and incubated overnight.The test compounds were added.The Coulter DNA Prep reagents kit (Cat.No.: PN 6607055) from Beckman Coulter (Fullerton, CA) was used and assayed as described previously (23).Analysis was done on a Beckman-Coulter Epics FC500 analyzer using CXP software for acquisition and the ModFit LT v3.1 (Verity Software) for Cell Cycle Modeling.Experiments were repeated three times. ",
"section_name": "Cell cycle",
"section_num": null
},
{
"section_content": "The cell attachment assay (ECM205) was used to study the effects of VPA on cell attachment to different components, including fibronectin, vitronectin, laminin, collagen I and collagen IV (ECM205, Chemicon).The assay was performed after a 72-hour incubation according to the kit instructions (Chemicon).One ml of cells (1x10 5 cells/ml) was plated in 24-well plates and treated with or without the test compound-VPA.The treated cells were harvested with enzyme-free cell dissociation buffer and added to each well of strips that were pre-rehydrated with PBS buffer.The strips were incubated for 45-60 minutes at 37 o C in the incubator.The strips were washed 3 times, stained with 0.2% crystal violet for 5 minutes and washed 3-5 times.One hundred µl of solubilization buffer was added to each well and the absorbance at 570 nm was determined after the cell-bound stain was completely solubilized. The assays were normalized by cell proliferation assays. ",
"section_name": "Cell attachment",
"section_num": null
},
{
"section_content": "Cervical cancer is the leading cause of cancer death in women.Notch signaling is of importance in cervical cancer progression, with this signaling being up-regulated in its early stage and down-regulated in late stage.The Notch-mediated mechanism in this cancer is not clear yet.Our previous data showed that Notch activation could enhance for skolin-induced cAMP production (19).In this study, we evaluated the effects of Notch activation on cell growth and analyzed the involvement of cAMP/Ca 2+ signaling in Notch-mediated growth suppression. ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "We investigated the effects of Notch1 signaling on cervical cancer Hela cells.Via MTT assay, Hela-ICN1 cells with Notch1 activation were found to grow slowly compared to control Hela-GFP cells, with Notch-induced inhibitory rates being 52.4%, and with no significant difference in Hela-DNL1cells (knockdown of Notch signaling) (Fig. 1).Notch signaling inhibitors DBZ and DAPT had no effect at the tested concentrations (data not shown).Furthermore, we did transient transfection with the active forms of all four Notch receptors (ICN1, ICN2, ICN3, ICN4) and found that all four ICNs could induce cell growth suppression, with ICN1 and ICN2 displaying more significant effects on Hela cell growth.As shown in Fig. 2, the effects are dose-dependent, with the inhibitory rates being 25.3% (ICN1: 200ng), 40.2% (ICN1: 400ng), 36.9% (ICN2: 200ng), 50.3 % (ICN2, 400ng), 25.5% (ICN3: 200ng), 28.6% (ICN3, 400ng), 15.9% (ICN4: 200ng) and 22.5% (ICN4, 400ng).These data support that Notch signaling serves as a tumor suppressor in cervical cancer Hela cells. Further, FACS analysis was used to evaluate Notch1-mediated cell cycle progression.Notch1 induced cell cycle arrest at phase G1 (Hela-ICN1), with a rate of 62% compared to 47% of the control (Hela-GFP) (Fig. 3).Meanwhile, FACS analysis showed that the Hela-ICN1 cell debris was 11% compared to 4% of Hela-GFP cell debris. Cell attachment is a critical step for cell migration and tumor metastasis.Also in our previous study, we observed Notch signaling activation could induce cervical cancer cell morphological changes in Hela-ICN1 cells.Thus, we did cell attachment assays to assess the effects of Notch signaling on cell attachment.We first tested the attachment of cervical cancer Hela cells to the extracellular matrix (ECM) components that included fibronectin, vitronectin, laminin, collagen I and collagen IV and found that cells strongly attached to laminin, but had little or no attachment to the others.Then, we found that Notch1 signaling activation, via transient transfection of ICN1, showed no significant effect on the attachment of cervical cancer Hela cells to the ECM component laminin (ECM103), but THBS1, upregulated by ICN1, was observed to increase cell attachment (Fig. 4).Compared to the control (Hela-GFP), ICN1's over-expression induced cell growth arrest in Hela-ICN1 cells (Notch signaling activation), with an inhibitory rate of 52.4%, but there is no significant effect in Hela-DNL cells (Notch signaling knockdown).A, cell proliferation assay; B, cell colony assay.Asterisk (*) shows P value being less than 0.05. ",
"section_name": "Activated Notch signaling acts as a tumor suppressor",
"section_num": null
},
{
"section_content": "Our previous study showed that Notch1 activation extremely enhanced for skolin-induced production of the second messenger cAMP that transduces intracellular signaling, and it also regulated somatostatin signaling that belongs to G protein-coupled receptor signaling.This implied that Notch signaling might be involved in signaling pathways associated with cAMP and GPCRs.Thus, via PCR arrays, we investigated the effects of Notch1 activation on the genes associated with cAMP/Ca 2+ signaling and GPCR signaling.We found that Notch1 activation in Hela-ICN1 cells regulated the expressional changes of certain genes that are related to CRE (cAMP responsive element), SRE(serum response element), Ca 2+ and other cAMP-coupled G protein signaling. ",
"section_name": "Notch1 signaling activation regulates gene expression",
"section_num": null
},
{
"section_content": ").There are no significant effects of genes such as ICN1, NR4A2, vinculin, THBS1, Twist1 on ECM103.However, THBS1 enhanced cell adherence to laminin, with a rate of 58.1%, and asterisk (*) shows P value < 0.05. As shown in Table 1, ICN1 induced an increase in such genes as vinculin (VCL) (5.2 fold), SCG2 (8.8 fold), ADRB2 (4.1 fold), SST (203 fold), SSTR2 (20.8 fold) and THBS1(TSP1) (60.7 fold), and a decrease of such genes as NR4A2 (-5 fold), SGK1(-1.5 fold), NPY(-3.8fold), ENO2 (-1.7 fold), CALR (-1.3 fold), PCK2 (-2.8 fold), CREB1 (-1.6 fold), CGA (-15.6 fold), NF1(-1.4fold), BRCA1(-1.5 fold), VEGFA (-1.82 fold) and RB1 (-2.8 fold), (Table 1) and had no effect on genes with CRE/SRE sequences in the promoters or belonging to GPCRs such as FGF6, IL-2, IL-6, GCG2, DRD1, PDPK1 andOPRD1 (data not shown).Certain other genes (and some cancer-related genes) were observed to be modulated by Notch1 activation, with an ICN1-induced increase of Notch target genesHES1, p63, p21 and Twist1, and a decrease of c-Myc.The expression of certain of these genes such as NR4A2, HES1, p63, p21 and c-Mycin Hela-ICN1 cells was confirmed at the protein level by Western blot analysis (Fig. 5A).Next, we investigated the effects of some chosen genes on cell growth and their involvement in Notch1-mediation signaling pathways (discussion below). ",
"section_name": ". The effects of gene over-expression on cell-laminin adhesion (ECM103",
"section_num": null
},
{
"section_content": "We selected six Notch1-mediated genes including NR4A2, Vinculin, THBS1, p63 and Twist, and did transient transfection with the plasmids expressing those genes.We assessed the effects of these genes on cell growth by cell proliferation assay and found stimulative effects by NR4A2, with a rate of 34.7%, but with little or no significant effects on VCL, p63, THBS1 or Twist1 (Fig. 6).As described above, we observed that Notch1 activation, by over-expressing ICN1, reduced cell-laminin attachment.We also investigated the effects of NR4A2, VCL, THBS1 and Twist1 on Hela cell attachment.By cell attachment assay (ECM103), we found that THBS1 induced an increase of the cell-laminin attachment, with a rate of 58.1%, with the other three, NR4A2, VCL and Twist1, having no significant effects (Fig. 4).These findings indicate that different mechanism are involved in cell proliferation and cell attachment. Via FACS analysis, we further found that NR4A2 reduced cell apoptosis.Cells were continuously cultured for 2 days without changing medium after transfection with the transfection agent Lipo-2000.This would result in some cell death.We further did apoptosis assays and found that there was an apoptotic rate 89.4% (apoptosis and necro-apoptosis together) in the control group with the control pCCL vector, but the apoptotic rate dropped to 64.4% in the NR4A2-treated group and with an increased rate of visible cells (22.3%) (Fig. 7). Based on the results above, we hypothesized that NR4A2 might play an oncogenic role and thus further analyzed the effects of NR4A2 activation on the oncogene c-Myc, tumor suppressors p21 and p63, and the Notch target gene HES1.We observed thatNR4A2 activation induced a decrease of p21, p63, and HES1, with a slight increase of c-Myc (Fig. 5B), indicating Notch signaling and NR4A2 signaling reversely co-modulate downstream signaling while Notch plays the tumor-suppressive role and NR4A2 plays the oncogenic role. ",
"section_name": "NR4A2 increases cell proliferation and THBS1 increases cell attachment",
"section_num": null
},
{
"section_content": "NR4A2 may be involved in ICN1-mediated effects on cervical cancer cells.We further tested this hypothesis by transient transfection of NR4A2 in Hela-ICN1cells (Notch1 activation), with Hela-GFP cells as the control.We found that NR4A2reversed ICN1-induced cell growth arrest.As shown in Fig. 8A, ICN1 decreased cell growth with an inhibition rate of 52%, NR4A2 reduce the ICN1-induced inhibitory rate to 44%.Also, via Western blot analysis, we found that NR4A2 reversed an ICN1-induced increase of p63 (Fig. 8B), but not of p21 and c-Myc, indicating that NR4A2 and Notch signaling share a common signaling involvement with p63 and that Notch1 induces cell growth arrest partly via regulating the downstream NR4A2/p63 signaling pathway. ",
"section_name": "NR4A2 reverses ICN1-induced cell growth suppression via regulating p63 signaling",
"section_num": null
},
{
"section_content": "The role of Notch signaling in cervical cancer is not clearly determined.The levels of Notch signaling in cervical cancer seem to be stage-specific.Activated Notch1 at high levels, induces apoptosis and cell growth arrest through increasing the expression of p52, p21 and others (6,12).However, Notch signaling in this cancer is dysfunctional and controversial (16,17,21).Thus, a more complete understanding of the molecular mechanisms of this Notch pathway in cervical cancer will lead to the development of novel therapeutic strategies.In our previous study in cervical cancer, Notch1 activation could induce cell growth arrest and tumor suppression, identical to other reports. In the present study, we did assays to confirm the function of Notch in cervical cancer cells.Our assays showed that the knockdown of Notch signaling via over-expressing dominant-negative mastermind-like1 (DNL1, or dnMAML1) did not significantly affect cervical cancer Hela cell growth.However, we confirmed the inhibitory effects of Notch1 activation (16,19,20), and identified the similar inhibitory effects via the activation of the other three Notch receptors (ICN2, 3 and 4).We also found that activated Notch signaling stimulated the somatostatin signaling that blocks cAMP signaling, and enhanced for skolin-induced cAMP production (19).Thus, we hypothesized that cAMP signaling might be involved in Notch-mediated signaling networks. cAMPis one well-known second messenger and is involved in various biological activities.cAMP signaling modulates cell functions and the associated signaling pathways.Our previous studies showed that Notch induced an increase of SST and SSTR2, with promotors' containing a CRE (cAMP responsive element) site (19).Meanwhile, we found that Notch modulated the expression of certain genes associated with cAMP/Ca 2+ signaling, especially the genes with promotors containing CRE, SRE and Ca 2+ elements and being responsive to cAMP or Ca 2+ .As shown in Table 1, Notch induced an increase of such genes as SST, SSTR2, THBS1 and VCL, and a decrease of such genes as NR4A2, STAT3 and CGA.Further, we evaluated the three CRE-containing genes VCL, THBS1 and NR4A2, tumor suppressor p63 and cell differentiation factor Twist1, for their effects on cell growth and cell adhesion.The nuclear receptor NR4A2 was observed to stimulate cell proliferation with others having no significant effects.In the cell adhesion assay, NR4A2 did not work, but the angiogenesis regulator THBS1increased Hela cells adhesion to laminin.NR4A2, acting as a transcription factor, induced cell differentiation and maintained dopaminergic neurons (24,25).THBS1 is an adhesive glycoprotein and affects cell adhesion and cell migration (26,27).Activated Notch signaling attenuates HPV-induced cervical cell transformation while its inactivation promotes cell transformation and carcinogenesis (2,7,28).Notch might partly suppress cell growth via attenuating NR4A2 and affect cell transformation and tumor metastasis via activating THBS1. In summary, the identification of the correlation of Notch signaling and these factors in cervical cancer cells may support these findings indicating that targeting these crosstalk events likely will provide valuable paradigms to currently available clinical interventions targeting cervical cancer.Meanwhile, cAMP signaling may play a critical role in Notch-mediated cell functions via modulating the pathways involved with such genes as NR4A2 and THBS1.The precise mechanisms need to be identified and could be helpful in developing targeted drugs against cervical cancer. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "The authors gratefully acknowledge the financial supports from Shanghai Science and Technology Committee of China (Grant No.14411972700), the financial supports from Tulane Peptide Research ",
"section_name": "Acknowledgement",
"section_num": null
},
{
"section_content": "Fund and Open Project Funding from State Key Laboratory of Genetic Engineering of Fudan University Open Project Funding. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors have declared that no competing interest exists. ",
"section_name": "Competing Interests",
"section_num": null
}
] |
10.1186/s12890-020-1154-6
|
LncRNA FEZF1-AS1 promotes non-small lung cancer cell migration and invasion through the up-regulation of NOTCH1 by serving as a sponge of miR-34a
|
<jats:title>Abstract</jats:title><jats:sec> <jats:title>Background</jats:title> <jats:p>The involvement of lncRNA FEZF1-AS1 has been analyzed in many types of cancers, while its roles in non-small cell lung cancer (NSCLC) remains unclear. We then explored the role of FEZF1-AS1 in NSCLC.</jats:p> </jats:sec><jats:sec> <jats:title>Methods</jats:title> <jats:p>qPCR and western blot were performed to measure gene expression. FEZF1-AS1, miR-34a, and NOTCH-1 were overexpressed to analyze the relationship between them. Transwell assays were performed to analyze the effects of transfections on cell invasion and migration.</jats:p> </jats:sec><jats:sec> <jats:title>Results</jats:title> <jats:p>FEZF1-AS1 was up-regulated in NSCLC patients. Increased expression levels of FEZF1-AS1 were observed with the increase in clinical stages. Bioinformatics analysis showed that miR-34a can bind with FEZF1-AS1. In NSCLC tissues, NOTCH-1 and FEZF1-AS1 were positively correlated. In NSCLC cells, over-expression of FEZF1-AS1 resulted in up-regulated expressions of NOTCH-1, while miR-34a over-expression mediated down-regulated expressions of NOTCH-1. In addition, FEZF1-AS1 and miR-34a did not alter each other, while bioinformatics analysis showed that miR-34a can bind FEZF1-AS1. Analysis of cell migration and invasion showed increased cell invasion and migration rates after FEZF1-AS1 and NOTCH-1 over-expression. MiR-34a played the opposite role and reduced the effects of FEZF1-AS1 over-expression.</jats:p> </jats:sec><jats:sec> <jats:title>Conclusions</jats:title> <jats:p>FEZF1-AS1 promoted NSCLC cell migration and invasion through the up-regulation of NOTCH1 by serving as a sponge of miR-34a.</jats:p> </jats:sec>
|
[
{
"section_content": "For decades, lung cancer ranks the first place for both mortality and incidence among all cancers [1].In 2018, 2,093,876 new cases of lung cancer were diagnosed and 1,761,007 deaths caused by lung cancer were recorded [1,2].NSCLC accounts for more than 85% of all cases [3,4].Moreover, most NSCLC patients are diagnosed with tumor metastasis to other parts of the body and the 5-year overall survival rate is as low as 10% [5,6].Therefore, novel therapeutic regimens are needed. Smoking habit is closely correlated with the incidence of NSCLC [7], while NSCLC also affects non-smokers [8].As another factor, genetic alterations also contribute to the occurrence and progression of NSCLC [9].Notch homolog 1 (NOTCH-1) plays critical roles in cancer development by inducing epithelial-mesenchymal transition, promoting cell stemness and enhancing cell invasion [10,11].Some miRNAs, such as miR-34a, targets NOTCH-1 to inhibit cancer progression [12].Through different ways, such as miRNA sponge or methylation regulator, lncRNAs interact with miRNAs to participate in diverse biological processes [13].LncRNA FEZF1-AS1 has been reported to promote the progression of several cancers, such as lung cancer [14,15], while the functionality remains to be further elucidated.In this study, we performed RNA-RNA interaction prediction using IntaRNA 2.0 and we found that miR-34a may bind FEZF1-AS1.Therefore, we will be focused to analyze the relationship between FEZF1-AS1 and miR-34a in NSCLC and the effects on NOTCH-1. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "In this study, we selected 39 male and 27 female NSCLC patients (age: 35 to 67 year-old; mean age: 51.1 ± 5.6 year-old) from 177 cases of NSCLC admitted to The Third Affiliated Hospital of Guangxi Medical University between March 2016 and April 2019.This study was approved The Third Affiliated Hospital of Guangxi Medical University Ethics Committee.Inclusion criteria: 1) newly diagnosed NSCLC; 2) no therapies were initiated.Exclusion criteria:1) complicated with other clinical disorders; 2) recurrent NSCLC; 3) therapies were initiated; 4) previous history and/or family history of malignancies.66 NSCLC patients were informed with the experiment design and potential publication of this paper, and all 66 patients signed informed consent.Clinical staging was performed using AJCC system.Based on clinical data, the 66 patients included 10, 12, 23 and 21 cases at stage I-IV, respectively. ",
"section_name": "NSCLC patients",
"section_num": null
},
{
"section_content": "Fine needle aspiration was performed on all patients to collect paired NSCNC and non-tumor specimens.Following histological confirmation, all tissue samples were immediately subjected to RNA isolations and following experiments. H1993 human NSCLC cell line (ATCC, USA) was used in this study.Cell culture medium was prepared by mixing RPMI-1640 Medium (90%) with FBS (10%).A 95% humidity and 5% CO 2 incubator was used to cultivate cells. ",
"section_name": "NSCLC tissue samples and cells",
"section_num": null
},
{
"section_content": "Negative control (NC) miRNA and the mimic of miR-34a, as well as vectors (pcDNA3.1)expressing NOTCH-1 and FEZF1-AS1, were from GenePharma (Shanghai, China).Vector (1 μg) or miRNA (40 nM) was transfection into 4 × 10 6 H1993 cells using lipofectamine 2000 (GenePharma).To perform NC experiments, H1993 cells were transfected with empty pcDNA3.1 vector or NC miRNA.To perform Control (C) experiment, cells without transfections were cultivated until the end of transfections.Cells were cultivated for further 48 h after transfections before use. ",
"section_name": "Vectors, miRNA mimic and transfections",
"section_num": null
},
{
"section_content": "H1993 cells were harvested and counted.All tissue samples (about 0.012 g) were ground into powder in liquid nitrogen.Total RNAs in 4 × 10 5 cells or tissue samples were extracted using Ribozol (Sigma-Aldrich, USA).To harvest all types of RNAs (such as miRNAs), RNAs were precipitated and washed using 85% ethanol. ",
"section_name": "RNA extractions",
"section_num": null
},
{
"section_content": "DNase I was used to incubate with RNA samples for 90 min at 37 °C to achieve genomic DNA removal.In order to measure the expression levels of FEZF1-AS1 and NOTCH-1 mRNA, total RNAs were reverse transcribed into cDNA and qPCRs were performed with GAPDH as internal control.Expression of miR-34a was determined using All-in-One™ miRNA qRT-PCR Detection Kit (Genecopoeia).U6 was included to sever as the Fig. 1 FEZF1-AS1 was up-regulated with an increase in clinical stages of NSCLC patients.QPCR and paired t-test were used to measure and compare expression levels of FEZF1-AS1 between two types of tissues (NSCLC vs. non-tumor) (a).ANOVA (one-way) and Tukey test were used to compare expression levels of FEZF1-AS1 in NSCLC tissues among different clinical stages (b).Data of 3 replicates were presented, *, p < 0.05 endogenous control of miR-34a.Ct values of three technical replicates were normalized using 2 -ΔΔCT method. ",
"section_name": "RT-qPCR",
"section_num": null
},
{
"section_content": "The interaction between miR-34a and FEZF1-AS1 was predicted using an online program named IntaRNA 2.0 (http://rna.informatik.uni-freiburg.de/IntaRNA/Input.jsp).All parameters were the default.FEZF1-AS1 was set to be the long sequence and miR-34a was set to be the short sequence. ",
"section_name": "RNA interaction prediction",
"section_num": null
},
{
"section_content": "At 24 h post-transfection, H1993 cells were counted and total proteins in 4 × 10 5 cells were extracted using RIPA solution (GenePharma).Total proteins were denatured in boiling water for 5 min.Proteins were then separated by 10% SDS-PAGE gel, transferred to PVDF membranes, and PBS (5% non-fat milk) was used to block membranes for 1 h at 24 °C.After that, rabbit anti-NOTCH-1 (1: 1200, ab52301, Abcam) and GAPDH (1: 1200, ab37168, Abcam) primary antibodies were used to incubate the membranes for 18 h at 4 °C.After that, further incubation with IgG H&L (IgG) (1:1000; ab6721; Abcam) secondary antibody was performed for 2 h at 24 °C.Signal development was performed by incubating membranes with RapidStep™ ECL detection reagent (EMD Millipore) for 5 min.Signals were processed using Image J v1.47 software. ",
"section_name": "Western blot",
"section_num": null
},
{
"section_content": "At 24 h post-transfection, H1993 cells were counted and transferred to upper Transwell chamber (4 × 10 3 cells in 0.1 ml serum-free medium per well).Matrigel (Millipore)coated membranes were used in invasion assay and uncoated membranes were used in migration assay.In both cases, lower chamber was filled with medium supplemented 20% FBS.Under aforementioned conditions, cells were cultivated for 12 h and 0.5% crystal violet (Sigma-Aldrich) was used to stain cell for 20 min at room temperature.An optical microscope was used to observe stained cells. ",
"section_name": "Transwell assays",
"section_num": null
},
{
"section_content": "Mean ± SD values of 3 biological replicates were calculated.Paired t test was used to compare paired tissues.ANOVA Tukey's test was used to compare multiple groups.Linear regression was used for correlation analysis.Patients were first grouped in to high and low (n = 33) FEZF1-AS1 level groups.The cutoff value was the median FEZF1-AS1 expression level in NSCLC.Correlations between expression levels of FEZF1-AS1 and patients' clinical data were analyzed by Chisquared test.p < 0.05 was statistically significant. ",
"section_name": "Data analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Analysis of RT-qPCR data revealed significantly higher expression levels of FEZF1-AS1 in NSCLC tissues compared to non-tumor tissues (Fig. 1a, p < 0.05).Expression levels of FEZF1-AS1 in NSCLC tissues among different clinical stages were compared.With the increase in clinical stages, significantly increased expression levels of FEZF1-AS1 were observed (Fig. 1b, p < 0.05).Chi-squared test showed that expression levels of FEZF1-AS1 in NSCLC tissues were significantly correlated with patients' clinical stage and tumor grades, but not patients' gender, age, subtypes and drinking and smoking habits (Table 1). ",
"section_name": "FEZF1-AS1 was up-regulated with an increase in clinical stages of NSCLC patients",
"section_num": null
},
{
"section_content": "Analysis of RT-qPCR data revealed significantly higher expression levels of NOTCH-1 mRNA in NSCLC tissues compared to non-tumor tissues (Fig. 2a, p < 0.05).Correlation analysis showed that, across NSCLC tissues, FEZF1-AS1 and NOTCH-1 were significantly and positively correlated (Fig. 2b, p < 0.05). ",
"section_name": "NOTCH-1 mRNA was up-regulated and positively correlated with FEZF1-AS1",
"section_num": null
},
{
"section_content": "The positive correlation between FEZF1-AS1 and NOTCH-1 mRNA indicated possible interaction between them, while miR-34a can target NOTCH-1.Therefore, the relationship between them was predicted.As shown in Fig. 3a, miR-34a can strongly bind to FEZF1-AS1.To further investigate the mechanism, FEZF1-AS1 or NOTCH-1 expression vector, or miR-34a mimic, was transfected into H1993 cells.QPCR showed that expression levels of miR-34a, FEZF1-AS1 and NOTCH-1 were significantly upregulated compared to C and NC groups at 24 h posttransfection (Fig. 3b, p < 0.05).Moreover, over-expression of miR-34a and FEZF1-AS1 did not significantly alter the expression of each other (Fig. 3c, p > 0.05).In contrast, over-expression of FEZF1-AS1 resulted in up-regulated NOTCH-1, while miR-34a over-expression mediated down-regulated NOTCH-1 and the reduced effects of FEZF1-AS1 over-expression (Fig. 3d, p < 0.05).Please check Supplementary Figure 1 for representative images of Western blot results. FEZF1-AS1 promoted the invasion and migration of NSCLCs through the axis of NOTCH-1 and miR-34a Invasion (Fig. 4a) and migration (Fig. 4b) of H1993 cells in different transfection groups were analyzed by Transwell assay.Compared to C and NC groups, cells with NOTCH-1 or FEZF1-AS1 expression vector transfection exhibited significantly increased cell invasion and migration.MiR-34a played the opposite role and reduced the effects of FEZF1-AS1 over-expression (p < 0.05). ",
"section_name": "FEZF1-AS1 sponges miR-34a to up-regulate NOTCH-1",
"section_num": null
},
{
"section_content": "We investigated the roles of FEZF1-AS1 in NSCLC.Our study revealed that FEZF1-AS1 was up-regulated in NSCLC.In addition, FEZF1-AS1 may sever as an endogenous sponge of miR-34a to up-regulate NOTCH-1 in NSCLC cells, thereby increasing the invasion and migration of cancer cells.The function of FEZF1-AS1 has been investigated in many types of cancers.For instance, FEZF1-AS1 is up-regulated in colorectal cancer and can regulate PKM2 signaling to promote cancer cell metastasis and proliferation [14].In multiple myeloma, FEZF1-AS1 is also overexpressed and over-expression of FEZF1-AS1 mediates the growth of tumor [16].In a recent study, He et al. found that FEZF1-AS1 was over-expressed and is correlated with epithelial-mesenchymal transition [15].Consistently, this study also reported the up-regulation of FEZF1-AS1 in NSCLC, and the over-expression of FEZF1-AS1 resulted in the increased invasion and migration rates of NSCLC cells.Our study further the role of FEZF1-AS1 as an oncogenic lncRNA in NSCLC. This study predicted the potential interactions between FEZF1-AS1 and miR-34a.It has been well established that miRNAs can regulate gene expression by direct cleavage or translation termination [17].However, over-expression experiments showed that miR-34a has no regulatory roles in the expression of FEZF1-AS1.Therefore, miR-34a is not likely to target FEZF1-AS1.Recent studies have showed that lncRNAs may mimic the targets of miRNAs, thereby sponge miRNAs to attenuate their effects on silencing downstream genes.It is known that miR-34a can target NOTCH-1 in glioblastoma to suppress cancer growth.In this study we also observed the downregulated NOTCH-1 at both mRNA and protein levels.Therefore, miR-34a may also target NOTCH1 in NSCLC.In addition, FEZF1-AS1 over-expression resulted in the up-regulated NOTCH-1 at both mRNA and protein levels.Therefore, our data support the speculation that FEZF1-AS1 may sponge miR-34a to up-regulate NOTCH-1, thereby promoting the invasion and migration of cancer cells.However, the mechanism remains to be further explored. Our data may suggest FEZF1-AS1 as a potential therapeutic target for NSCLC.However, our study did not include clinical trials.Therefore, future studies are needed to perform clinical studies or animal model studies to further confirm our conclusions. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "In conclusion, our study confirmed the oncogenic role of FEZF1-AS1 in NSCLC and we proposed that FEZF1-AS1 may sponge miR-34a to up-regulate NOTCH-1 to promote cancer cell invasion and migration. ",
"section_name": "Conclusion",
"section_num": null
}
] |
[
{
"section_content": "Not applicable. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "Not applicable. ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "The analyzed data sets generated during the study are available from the corresponding author on reasonable request. ",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "Supplementary information accompanies this paper at https://doi.org/10.1186/s12890-020-1154-6. Additional file 1: Figure S1.Representative images of Western blot results.Original images here are of a biological replicate of Western blot.Data here is the same information as shown in Fig. 4c. Abbreviation NSCLC: Non-small cell lung cancer; lncRNAs: Long (> 200 nt) non-coding RNAs; NOTCH-1: Notch homolog 1 Authors' contribution SXH conception, interpretation of data, revised the manuscript, supervision.CJL, JFH, PHH, DCM, HLM acquisition, analysis of data, software, drafted the manuscript.All the authors have approved the submitted version, and ensured the questions related to the accuracy or integrity of any part of the work. The present study was approved by the Ethics Committee of The Third Affiliated Hospital of Guangxi Medical University.The research has been carried out in accordance with the World Medical Association Declaration of Helsinki.All patients and healthy volunteers provided written informed consent prior to their inclusion within 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": "Supplementary information",
"section_num": null
},
{
"section_content": "Supplementary information accompanies this paper at https://doi.org/10.1186/s12890-020-1154-6. Additional file 1: Figure S1.Representative images of Western blot results.Original images here are of a biological replicate of Western blot.Data here is the same information as shown in Fig. 4c. Abbreviation NSCLC: Non-small cell lung cancer; lncRNAs: Long (> 200 nt) non-coding RNAs; NOTCH-1: Notch homolog 1 Authors' contribution SXH conception, interpretation of data, revised the manuscript, supervision.CJL, JFH, PHH, DCM, HLM acquisition, analysis of data, software, drafted the manuscript.All the authors have approved the submitted version, and ensured the questions related to the accuracy or integrity of any part of the work. ",
"section_name": "Supplementary information",
"section_num": null
},
{
"section_content": "The present study was approved by the Ethics Committee of The Third Affiliated Hospital of Guangxi Medical University.The research has been carried out in accordance with the World Medical Association Declaration of Helsinki.All patients and healthy volunteers provided written informed consent prior to their inclusion within 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
}
] |
10.1186/1477-3155-5-4
|
Potential therapeutic application of gold nanoparticles in B-chronic lymphocytic leukemia (BCLL): enhancing apoptosis
|
B-Chronic Lymphocytic Leukemia (CLL) is an incurable disease predominantly characterized by apoptosis resistance. We have previously described a VEGF signaling pathway that generates apoptosis resistance in CLL B cells. We found induction of significantly more apoptosis in CLL B cells by co-culture with an anti-VEGF antibody. To increase the efficacy of these agents in CLL therapy we have focused on the use of gold nanoparticles (GNP). Gold nanoparticles were chosen based on their biocompatibility, very high surface area, ease of characterization and surface functionalization. We attached VEGF antibody (AbVF) to the gold nanoparticles and determined their ability to kill CLL B cells. Gold nanoparticles and their nanoconjugates were characterized using UV-Visible spectroscopy (UV-Vis), transmission electron microscopy (TEM), thermogravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS). All the patient samples studied (N = 7) responded to the gold-AbVF treatment with a dose dependent apoptosis of CLL B cells. The induction of apoptosis with gold-AbVF was significantly higher than the CLL cells exposed to only AbVF or GNP. The gold-AbVF treated cells showed significant down regulation of anti-apoptotic proteins and exhibited PARP cleavage. Gold-AbVF treated and GNP treated cells showed internalization of the nanoparticles in early and late endosomes and in multivesicular bodies. Non-coated gold nanoparticles alone were able to induce some levels of apoptosis in CLL B cells. This paper opens up new opportunities in the treatment of CLL-B using gold nanoparticles and integrates nanoscience with therapy in CLL. In future, potential opportunities exist to harness the optoelectronic properties of gold nanoparticles in the treatment of CLL.
|
[
{
"section_content": "There is increasing evidence that angiogenesis plays a critical role in the pathogenesis of human malignancies [1,2].Angiogenesis is an event that relies on the formation of vessels from preexisting vasculature that occurs in health and disease.Initially it was found that without new capillary formation there could not be significant tumor growth or metastasis to other organ sites.While the original evidence for this was based on the finding of tissue neovascularization, there have been significant advances delineating the presence of autocrine and/or paracrine pathways in both solid tumors and human leukemias [3,4].Hematological diseases with aberrant vascularization include; multiple myeloma, acute myeloid leukemia and more recently B-chronic lymphocytic leukemia (CLL).These findings have led to the exciting possibility that strategies that undermine the angiogenic pathways could be used as non-overlapping methods of treatment for these diseases [5]. Initially the secretion of VEGF from malignant tumors was believed to be of primary importance in the development of neovascularization of the tumor involved tissue sites.This important biologic event was associated with more aggressive disease status.However more recently the paracrine role of VEGF has been modified to include autocrine pathway(s) that increase survival of malignant cells in both mouse and human tumor types [6].Interruption/ blockade of the VEGF pathway in those tumor cells has been shown to lead to cell death.To a great extent the level of interruption/blockade has been either to bind VEGF or to inhibit VEGFR-1 or VEGFR-2 [7,8].Importantly, ourselves and others have found that CLL B cells secrete VEGF and express the VEGF receptors; VEGFR-1, VEGFR-2 and Neuropilin-1 (NRP-1) [9].The VEGF based pathway appears to be important in the apoptosis resistance of CLL B cells.Thus we have found that culturing CLL B cells with receptor tyrosine kinase inhibitors or anti-VEGF antibodies (Avastin; bevacizumab) leads to increased levels of apoptosis.However, significantly high amount of the antibody was required to have a moderate effect in the apoptosis.In order to enhance the efficacy of agents such as anti-VEGF antibodies we have conducted initial studies utilizing delivery of these antibodies via conjugated gold nanoparticles.The primary rationale for selecting gold nanoparticles is their biocompatibility, very high surface area (large amount of drugs can be loaded), ease of characterization and surface modification (i.e.organic molecules such as drugs, peptides, antibodies, etc. can be easily attached to gold nanoparticles) [10].This report details our initial work with anti-VEGF (AbVF) conjugated to gold nanoparticles in comparison to naked anti-VEGF antibody or gold nanoparticles alone in the modulation of the apoptotic status of CLL B cells. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Results and discussion",
"section_num": null
},
{
"section_content": "Gold nanoparticles were synthesized according to standard wet chemical methods using sodium borohydride as a reducing agent [11][12][13].Characteristic surface plasmon resonance (SPR) band of gold nanoparticles was observed in the UV-Visible spectrum, confirming the presence of spherical gold nanoparticles (Figure 1a).TEM micrographs showed spherical gold nanoparticles of approximately 4 nm were formed by this method (Figure 1b).Size distribution analysis clearly showed that nearly 90% of the particles reside within 4 nm size range (Figure 1c).Gold nanoparticles obtained by this method were centrifuged at 13,000 rpm for 45 min at 10°C.The loose pellet at the bottom was collected and analyzed for gold content using inductively coupled plasma (ICP) analysis.The concentration of gold was found to be 200 µg/ml.The gold nanoparticles obtained after ultracentrifugation was filtered through 0.22 µM filter paper and UV-irradiated for 15 minutes before their use in the apoptosis assay as control for Au-AbVF. The decision to establish feasibility of anti-VEGF antibody conjugation to gold nanoparticles was based on our earlier observations that anti-VEGF antibodies alone could induce apoptosis of CLL B cells [14].However we wished to develop maneuvers that would enhance the ability of the anti-VEGF antibody to kill CLL B cells.Attachment of VEGF antibody (AbVF) to gold nanoparticles was done according to published literature and monitored using UV-Visible spectroscopy (Shimadzu, UV2401 PC) because the SPR is very sensitive to surface modification of the gold nanoparticles.An increase in absorbance of gold nanoparticles with a concomitant red shift in the λ max was also observed as reported earlier [13].The increase in absorbance and red shift in the λ max indicates the perturbation of the electrical double layer present around the gold nanoparticles on the addition of AbVF and confirms its attachment on the gold nanoparticles [13].Finally, the concentration of AbVF on gold nanoconjugates and its nature of bonding with gold nanoparticles were determined using thermogravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS).For TGA, 150 ml gold nanoparticles were incubated with 600 µg of AbVF.After 1 h, the nanoconjugates were centrifuged at 25000 rpm for 1 h, freeze-dried overnight and analyzed using TGA. Figure 2a and 2b describe the TGA profile of Au-AbVF nanoconjugates.Figure 2a clearly shows a total weight loss of 12% spanning over three distinct steps of weight losses at 200°C, 250°C and 375°C.The graph of derivative weight loss over temperature (Fig. 2b) shows three distinct maxima of weight losses at nearly 240°C, 320°C and 400°C.A weight loss of 13.3% should be observed according to theoretical calculation (where 1 ml gold nan- oparticles solution containing 26 µg of gold was incubated with 4 µg of AbVF).Therefore, a total weight loss of 12% accounts for 90% of the AbVF used initially for attachment was actually bound to gold nanoparticles.Three different weight losses at three different temperatures indicate that there are, at least, three different modes of bonding between AbVF and gold nanoparticles.Gold is known to bind strongly with organic molecules containing thiols and amines groups [18].The formation of selfassembled monolayers of organothiols on gold surfaces has been attributed to its ability to bind thiolates covalently [16,17].Since the discovery of immunogold labeling in 1971 by Faulk and Taylor, a great deal of information is known about the nature of bonding between gold nanoparticles and antibodies [18].It is now well recognized that there are, at least, three separate but dependent phenomenon that might explain the bonding of AbVF to gold nanoparticles; i) electrostatic attraction of negatively charged gold nanoparticles with positively charged protein molecules, ii) covalent bonding between the thiols/amine groups present within the amino acids in the antibody and the gold nanoparticles, and iii) hydrophobic interaction between proteins and gold nanoparticles [19].Our data suggests that after being electrostatically attracted to gold nanoparticles, AbVF (having a lysine and cysteine residues) binds covalently to the gold nanoparticles through the thiol/amine groups.The small amount of weight losses at relatively lower temperature is suggestive of a weaker interaction between gold and the antibody and we speculate it to be hydrophobic in nature.A second weight loss at higher temperature (320°C) indicates another distinctly different bonding mode that needed much higher energy to desorb/decompose the antibody from the nano-gold surfaces.The maximum weight loss at this temperature is most likely due to the presence of gold-sulfhydryl/goldamine bond where the antibody is held to the nano gold surface through covalent interaction between the gold nanoparticles and cysteine/lysine residues present in the antibody [20].A third weight loss at the even higher temperature (400°C) was suggestive of the presence of a third complex mode of attachment of the antibody to gold nanoparticles that requires higher energy to desorb/decompose from the nanogold surfaces. ",
"section_name": "Synthesis of gold nanoparticles and conjugation with anti-VEGF antibody",
"section_num": null
},
{
"section_content": "The nature of bonding between gold and AbVF was further supported by X-ray photoelectron spectroscopy (XPS).A single gold (Au 4f7/2) peak at 83.2 eV with a spin orbit coupling of 3.7 eV was observed in the drop-coated conjugates.These data clearly demonstrate that all of the Au +3 ions used in the process were reduced to Au 0 by sodium borohydride (Fig. 2c) [11,21].Two weak sulfur peaks were also observed.The presence of two sulfur peaks at 162.7 and 167.1 eV represents two chemically distinct sulfur species (data not shown).We observed similar sulphur peaks from gold conjugates containing VEGF165, a growth factor [11].This is not unexpected because both the VEGF165 and AbVF are proteins and it is reasonable to assume that they use similar chemical entities such as cysteine or lysine residues to bind to gold nanoparticles.The peak at 162.7 eV can be assigned to goldthiolate bond and peaks at higher binding energy (BE) to sulfones, an oxidized sulfur species.The origin of this sulfone peak may arise due to aerial oxidation of the sulfur during sample preparation [11].Unbound thiol peaks normally appear at 164 eV [21].The nitrogen 1s peak at 399.6 eV (Fig 2d) is likely due to unionized, non-protonated nitrogen [22].This is in agreement with earlier studies reported on the adsorption of proteins/amino acids or amines on gold surfaces [22,23].Therefore, we speculate that AbVF may bind to gold nanoparticles through -NH 2 functionalities via pseudo-covalent interaction.Hence, we infer from the XPS and TGA studies that AbVF binds to gold nanoparticles through sulfur and/or nitrogen present in the cysteine/lysine residues in the antibody.Following the establishment of a protocol that we could reliably generate and characterize gold nanoparticles conjugated to AbVF we then explored the ability of this conjugate to alter leukemic B cell apoptosis.For the apoptosis experiments, 150 ml gold nanoparticles were incubated with 600 µg of AbVF and purified through ultracentrifugation as described above and then used for the study after UV-Irradiating the particles for 20-30 minutes.Exactly the same amount of gold nanoparticles (Au) and AbVF present in the respective doses of Au-AbVF were separately used as controls.It is important to mention here that in the case of AbVF and Au-AbVF treated groups, the doses are always referred to based on the amount of AbVF present in the nanoconjugates, e. g, 25 µg Au-AbVF corresponds to 25 µg AbVF present in the nanoconjugates.However, in the case of gold treated groups the doses of 1, 5 and 25 µg correspond to the exactly same amount of gold present in 1, 5, and 25 µg of Au-AbVF respectively. ",
"section_name": "UV-Visible spectroscopy and transmission electron micrograph of gold nanoparticles",
"section_num": null
},
{
"section_content": "It is also important to note here that there is hardly any preclinical model available for CLL-B studies.Therefore, studies with primary CLL-B cells (cells isolated directly from patient blood) are considered as a preclinical study.Figure 3 describes the dose dependent behavior of AbVF, Au and Au-AbVF in inducing apoptosis of CLL B cells isolated from 7 different patients.CLL B cells were incubated with AbVF, Au and Au-AbVF separately for 72 hours followed by apoptosis measurement using Annexin/PI analysis.Figure 3a describes the effect of different doses of AbVF in inducing apoptosis.Among the 7 samples, 3 samples (P3, P5 and P7) showed moderate apoptosis levels (~40%) in a dose dependent manner; maximum apoptosis was observed at highest dose (25 µg).For the remaining 4 samples, 2 showed no effect (Sample P4 and P6) and 2 showed only a partial apoptosis above baseline (~10%).Figure 3b describes the effect of gold nanoparticles alone in the induction of apoptosis in CLL-B cells.The amount of gold nanoparticles used is exactly the same as in the respective doses of Au-AbVF.From this figure it is clear that 3 (P1, P2, P3) samples responded to the exposure to gold nanoparticles alone with increases in apoptosis (~50-60% apoptosis was observed) in a dose dependent manner.Among the remaining 4 samples, 2 did not respond at all (P6, P7) and lesser levels of apoptosis induction were observed for the remaining 2 samples (P4, P5). Figure 3c describes the effect of Au-AbVF in the induction of apoptosis in CLL-B cells isolated from patients.From figure 3c it is evident that all the 7 samples responded more effectively to the gold-AbVF treatments in terms of apoptosis induction.Significant increases in apoptosis (~80%) in a dose dependent manner were observed in 4 out of the 7 CLL Thermogravimetric analysis (TGA) of Au-AbVF conjugates Figure 2 Thermogravimetric analysis (TGA) of Au-AbVF conjugates.For TGA, 150 ml gold nanoparticles were incubated with 600 µg of AbVF.After 1 h, the nanoconjugates were centrifuged at 25000 rpm for 1 h, freeze-dried overnight and analyzed using TGA; 2a) describes weight loss over temperature and 2b) derivative weight loss over temperature for the nanoconjugates.TGA analysis was done on purified and lyophilized nanoconjugates, 2c) X-ray photoelectron spectra of Au-AbVF conjugates.Core level BE of Au and 2d) core level BE of N. Dose dependent effect of AbVF, Au and Au-AbVF in the induction of apoptosis in CLL-B cells Figure 3 Dose dependent effect of AbVF, Au and Au-AbVF in the induction of apoptosis in CLL-B cells.Cells were treated with 1, 5 and 25 ug of AbVF/Au/Au-AbVF for 72 h followed by measurement of apoptosis using annexin PI.The % apoptosis was calculated after normalizing the apoptosis in the control cells (no treatment group) to zero; 3a) effect of AbVF to induce apoptosis in CLL-B cells; 3b) effect of Au nanoparticles only to induce apoptosis in CLL-B and figure 3c) effect of Au-AbVF to induce apoptosis in CLL B cells.In the case of AbVF treated group, the amount of AbVF used is exactly the same in the corresponding doses of Au-AbVF.Similarly, in case of gold treated group, the amount of gold nanoparticles used is exactly the same as in the corresponding doses of Au-AbVF. samples and a significant enhancement in the apoptosis was observed in all the samples compared to Au and AbVF controls.The apoptosis induction observed in the case of control gold nanoparticles (Au treatment group) on CLL B cells is not surprising as we have shown previously that the gold nanoparticles posses unique antiangiogenic properties [11][12][13].Recently, it has been reported that gold nanoparticles inhibit the function of heparin binding growth factors such as VEGF165, BFGF and posses anti-angiogenic properties.Therefore, gold nanoparticles alone without any modification can inhibit the function of some of the growth factors secreted by the CLL B cells and hence the induction of apoptosis [11,12]. Figure 4 describes the time course of the effect of AbVF, Au and Au-AbVF in the induction of apoptosis to CLL-B cells.CLL-B cells were treated with 5 µg/ml of AbVF either bound or non-bound to gold as well as gold controls separately for 24 h, 48 h and 72 h respectively (the amount of gold in the control Au-treated good is exactly the same here as in 5 µg of Au-AbVF).Figure 4a shows the effect of non-conjugated AbVF to induce apoptosis in CLL-B cells treated with 5 µg/ml of AbVF.After the respective time points, cells were analyzed for apoptosis using annexin/PI method.Figure 4a shows the effect of non-conjugated AbVF over time and a clear level of apoptosis induction over baseline was observed in 3 samples out of 7 (P5, P6, P7).However, no clear time dependent apoptosis was observed with the 5 µg/ml dose.Figure 4b describes the effect of Au nanoparticles on the time dependent induction of apoptosis of CLL-B cells (n = 7).Time dependent apoptosis was observed for P1 and P3 where maximum apoptosis was observed at 72 h, however P2, P4 and P7 did not respond with apoptosis induction to the nanogold particles.For P5 and P6, time dependent apoptosis was observed for first 48 h, however, no apoptosis was observed at 72 h.We believe these residual B cells were resistant to the nonconjugated Au nanoparticles.The degree of apoptosis observed from the treatment with gold nanoparticles alone is not unexpected due to the anti-angiogenic properties of gold nanoparticles, as described above.Figure 4c describes the effect of Au-AbVF in the induction of apoptosis in CLL B cells.With this conjugate time dependent apoptosis was observed with maximum apoptosis observed at 72 h for all CLL B cells.Importantly in the case of Au-AbVF treated samples for both experiments depicted in figure 3 and4, all the CLL B cell samples showed significantly higher induction in apoptosis (2-5 fold) compared to either the AbVF treatment group or gold treatment groups (Fig 4d).It is also important to mention here that control experiments with same doses of nanoconjugates used in CLL-B cell studies using peripheral blood mononuclear cells (PBMCs) isolated from normal healthy individual did not induce significant apoptosis.Only a less than 10% apoptosis was observed with the highest does of Au alone or AbVF or Au-AbVF whereas the lower doses did not induce any significant apoptosis. Various autocrine pathways in CLL B cells provide important survival advantages for these cells.We have already established that a VEGF autocrine pathway exists in CLL B cells [7].The evidence for this includes our finding that recombinant VEGF165 can rescue CLL B cells from both spontaneous and drug-induced apoptosis.The addition of VEGF to CLL B cells also resulted in significant increases in the antiapoptotic proteins Mcl-1 and XIAP.In addition we found that the two VEGF receptors (VEGF-R1 and VEGF-R2) on CLL B cells are spontaneously phosphorylated.The combination of VEGF-induced increases in anti-apoptotic proteins combined with the finding of phosphorylated VEGF receptors strongly suggested that a VEGF-based pathway is linked to CLL Bcell survival.We consistently found that the anti-apoptotic protein Mcl-1 was increased when CLL B cells were exposed to exogenous VEGF165 [3].To find out the mechanism of more effective induction in apoptosis in CLL-B cells by Au-AbVF conjugates, we looked at the levels of Mcl-1, PARP, BcL-2 and caspase3 in CLL-B cells treated with different doses of Au, AbVF and Au-AbVF.Figure 5, shows that CLL B cells have clearly detectable PARP cleavage, decrease in caspase-3, Mcl-1 and Bcl-2 for Au-AbVF exposed cells but not for Au control or AbVF alone.Thus we believe that the AbVF conjugated to gold nanoparticles are inducing apoptosis in the CLL B cells similar to what we have found with VEGF pathway blockade using bevacizumab alone, but Au-AbVF is more effective than naked antibody 2C3 (i.e.2-5 fold increase in induction of apoptosis).Previously, we have shown our ability to bind anti-angiogenic molecules and anti-cancer drugs on a single gold core in a \"spoke in a wheel\" fashion [13].This study details our ability to enhance the efficacy of anti-VEGF antibody, 2C3, when conjugated to gold nanoparticles with resultant significant enhancement in apoptosis of primary CLL B cells when compared to an anti-VEGF antibody or gold nanoparticles alone.The reason for such an enhanced activity of Au-AbVF towards apoptosis induction is currently unknown and a subject of future investigation, but we believe that enhancing efficacy of the antibodies and lowering of the doses when delivered as gold nanoconjugates will have tremendous implications in the treatment of CLL. ",
"section_name": "Impact of gold nanoparticles (Au), and gold nanoparticles conjugated with AbVF (Au-AbVF) and AbVF on CLL B cell apoptosis",
"section_num": null
},
{
"section_content": "It is also important to know the fate of the nanoparticles after the treatment.Do the cells internalize the particles or do the particles remain bound to the cell membrane?To address these issues transmission electron microscopy of the cells treated with gold nanoparticles alone and with Au-AbVF for 1 h is performed [24].The 1 h treatment duration was chosen based on previously reported literature on the internalization of gold nanoparticles as longer time points did not increase gold uptake nor does it alter the pattern of internalization.Figure 6 exhibits the internalization of Au-AbVF by primary CLL-B cells after 1 h treatment.All the features of early as well as late internalization were clearly visible.Nanoconjugates were found at the cell periphery (within uncoated tubules and vacuoles, Figure 6a,b).Gold nanoparticles were also detected within larger endocytic compartments of diverse mor-phology.These include peripherally both early and late endosomes and lysozomes (6c, 6d).Similar pattern of internalization was observed when the cells were treated with gold nanoparticles alone (Figure 6e-6h).However, in this case, the number of gold nanoparticles taken up by the cells was found to be much lower than the previous case and lots of aggregated gold nanoparticles were seen.Since, the focus of this paper is not to find out the endocytic pathway of gold nanoparticles/gold nanoconjugates by primary CLL-cells, so further detailed studies to find out the mechanisms of internalization was not per- ",
"section_name": "Fate of gold nanoparticles and its conjugates after treatment",
"section_num": null
},
{
"section_content": "Effect of time on the induction of apoptosis of CLL-B cells by AbVF, Au and Au-AbVF.Cells were treated with 5 ug/ml of AbVF/Au/Au-AbVF for 72 h.Apoptosis measurement was then done using annexin/PI and apoptosis was calculated as described above; 4a) effect of AbVF alone; 4b) effect of Au alone; 4c) effect of Au-AbVF alone and 3d) comparison of the efficacy of AbVF, gold and gold-AbVF, 4d) comparison of effect of AbVF, Au and Au-AbVF on the apoptosis of CLL-B cells.To compare the activity of AbVF either conjugated or free the amount of AbVF used as control is exactly the same amount of AbVF present in the nanoconjugate.Similarly, to compare the activity of Au and Au-AbVF the amount of Au used as control is exactly the same amount of Au present in the nanoconjugate.The quantitations of Au in the nanoparticles solution and in the purified nanoconjugates were determined using inductively couple plasma analysis (ICP).While we may be concerned about the possible toxicities of gold nanoparticles in human therapies, colloidal gold actually has a long history of use in humans [25,26].The therapeutic use of gold can be traced back to the Chinese in 2500 BC [10].They were the first to prepare and use red colloidal gold as the \"drug of longevity.\"Red colloidal gold is still in use today in India in the form of Ayurvedic medicine for rejuvenation and revitalization during old age under the name of Swarna Bhasma (\"Swarna\" meaning gold, \"Bhasma\" meaning ash) [27,28].Further evidence of gold nanoparticles as potential non-toxic agents comes from our murine preclinical model in which we show that intraperitoneal administration of gold nanoparticles to C3H mice does not cause any acute biochemical and/or hematological toxicities [13].In vitro, the nontoxic behavior of gold nanoparticles has also been addressed by other groups including ours [29][30][31]. Another recent report has shown in a preclinical mouse model, that doses of gold nanoparticles as high as 2.7 g/ kg body weights did not cause any significant toxicity in mice up to 1 year of study [32].Furthermore, light absorbing properties of gold nanoparticles has been exploited to inflict localized damage to cells at sublethal levels to transiently modify cellular functions [33].Gold-silica nanoshell has been used in nanoshellassisted photo-ther-mal therapy (NAPT) to kill tumor cells by near infrared light (NIR) [34].Gold-EGFR conjugates have been used to detect the cancer cells by exploiting the light scattering properties of gold nanoconjugates [35].This paper is unique in that it bridges the field of nanoscience and technology with the therapy in CLL and opens up the opportunity of utilizing the technological advances of nanoscience in the treatment of CLL. These findings in CLL reinforce the advantages of using gold-nanoparticle-based drug delivery system in human malignancies.They include; increased concentration of drug loading due to high surface area of the nanoparticles, the ability to load multiple drugs (including the targeting agent) on the nanoparticle and the important feature of achieving higher local drug concentrations with reduced systemic toxicity and enhanced efficacy.We continue to develop this nanoparticle system in order to not only exploit the use of gold nanoparticles as anti-angiogenic targeted molecules but to conjugate other known chemotherapeutic (i.e.fludarabine) or monoclonal agents (i.e.rituximab) to these nanoparticles as well.This delivery system has the potential to induce cell death in CLL B cells by not only interrupting the VEGF based survival pathway but also by delivering effective levels of chemoimmunotherapeutics to the cells.In future, potential opportunities exist to harness the size dependent optoelectronic properties of gold nanoparticles in the treatment of CLL. ",
"section_name": "Effect of time on the induction of apoptosis of CLL-B cells by AbVF, Au and Au-AbVF Figure 4",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "Blood was obtained from healthy donors or CLL patients who had provided written informed consent.The laboratory study was approved by the Mayo Clinic Institutional Review Board according to the regulations of the Declaration of Helsinki.All CLL patients had a confirmed diagnosis using the NCI working group definition [36].Patients in this cohort were from all Rai stages and had not been treated for at least 6 weeks prior to blood processing for this study.Peripheral blood mononuclear cells (PBMC) were separated from heparinized venous blood by density gradient centrifugation.To remove adherent cells, PBMC were suspended in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and incubated in plastic dishes at 37.1°C for 1 h prior to collection of non-adherent cells. To obtain at least 95% purity of CLL B cells, non-adherent cells were depleted of T cells by incubation with sheep erythrocytes.In addition, we also purified CLL B cells from magnetic bead columns.To do this, highly purified CD19 + B cells (≥95%) were obtained from PBMC by standard negative selection using a cocktail of subset-specific antibodies conjugated with magnetic beads (Miltenyi Biotech, Auburn, CA, USA).These purified CLL B cells were then either used immediately for the laboratory studies described below or cryopreserved in RPMI 1640, 20% Internalization of Au-AbVF and Au alone by primary CLL-B cells after 1 h incubation.6a) nanoparticles were seen at the cell periphery (within uncoated tubules and vacuoles); 6b) higher magnification image of 6a; 6c and 6d) showing the internalized particles in different endocytic compartments.Figure 6e to 6h show the internalization of Au nanoparticles alone by primary CLL-B cells after 1 h incubation.6e) Nanoparticles were seen at the cell periphery (within uncoated tubules and vacuoles); 6f) higher magnification image of 6e; 6f) showing the internalized particles in multivesicular bodies; and 6g) higher magnification image of 6f. FCS, and 10% DMSO and stored in liquid nitrogen until use. ",
"section_name": "Patient selection and purification of lymphocytes",
"section_num": null
},
{
"section_content": "Primary CLL B cells and human splenic B cells obtained from controls were sorted by CD 19 antibody conjugated to magnetic beads (Miltenyi Biotec, Auburn, CA, USA) and were then cultured in serumfree AIM-V (Gibco BRL, USA) and RPMI (Biomol, USA) supplemented with 10% FCS, respectively.Cells were maintained at 37°C in an atmosphere containing 95% air-5% CO 2 (v/v). ",
"section_name": "Cell culture",
"section_num": null
},
{
"section_content": "Immunological reagents that recognize the following antigens were purchased from the indicated suppliers: Mcl-1 from BD Pharmingen (San Diego, CA, USA); Bcl-2 from Dako Corp (Carpinteria, CA, USA); XIAP from R&D Systems, Inc. (Minneapolis, MN, USA); β-actin from Novus Biologicals (Littleton, CO, USA).VEGF neutralizing antibody (2C3) was a kind gift from Pergerine Pharmaceuticals.The antibody (2C3) specifically blocks the interaction between VEGF and VEGFR-2 [37]. ",
"section_name": "Reagents",
"section_num": null
},
{
"section_content": "Gold nanoparticles were synthesized according to standard wet chemical methods using sodium borohydride as a reducing agent [11][12][13].Characteristic surface plasmon resonance (SPR) band of gold nanoparticles was observed in the UV-Visible spectrum, confirming the presence of spherical gold nanoparticles (Fig. 1a).TEM micrographs showed spherical gold nanoparticles of approximately 5 nm were formed by this method (Fig. 1b).The size distribution analysis of gold nanoparticles (after counting 500 individual particles) clearly shows that most of the gold nanoparticles (~90%) are in 4-5 nm range (Fig 1c). Binding of VEGF antibody to gold nanoparticles was done according to our previously published report [13].In brief, 150 ml of gold nanoparticles were incubated with 600 µg of AbVF for 1 h at room temperature under stirring.After 1 h, gold conjugates thus obtained were ultracentrifuged at 25000 rpm at 10°C for 1 h.The loose pellet obtained at the bottom was collected and UV-irradiated for 20-30 minutes before use in apoptosis assays.The binding was monitored using UV-Visible spectroscopy.UV-Visible spectrum was recorded on a Shimadzu model system (UV2401 PC) and the saturation concentration was determined. ",
"section_name": "Synthesis and characterization of gold nanoparticles and its nanoconjugates",
"section_num": null
},
{
"section_content": "For TGA analysis, 150 ml of gold nanoparticles were incubated with 600 µg of AbVF for 1 h at room temperature under stirring.After 1 h, gold conjugates were centrifuged at 25000 rpm at 10°C for 1 h.Both the supernatant and the loose pellet at the bottom were collected.The loose pellet obtained after ultracentrifugation was used for the quantification of AbVF attached on gold nanoparticles and also for apoptosis studies with CLL B cells described below.For TGA analysis, the pellet obtained after ultracentrifugation was lyophilized to obtain gold nanoconjugates in powder form.The lyophilized powder of nanoconjugates was used for TGA analysis.TGA was done using a TA Instruments Q500 thermal analyst.The TGA data were obtained in flowing nitrogen at a heating rate of 20°C/min.Amount of drugs attached onto gold nanoparticles were obtained from weight loss from the TGA curve as previously described [10]. Studying nature of bonding by X-ray photoelectron spectroscopy X-ray photoelectron spectroscopy (XPS) was performed to find out the nature of bonding between gold nanoparticles and the antibody.XPS was obtained on a PHI 5400 instrument using a Mg Kα Xray (1253.6 eV) anode source operated at 250 W, pressure was below 2 × 10 -9 torr.The electron pass energy on the hemispherical analyzer was set at 89.45 eV for survey scans and 17.9 eV for highresolution scans.The binding energy scale was referenced to that of C1s (285.0 eV).Samples were prepared by drop-coating anti-VEGF165 antibody conjugated gold solution on a clean silicon wafer and the drops were allowed to air dry [38]. ",
"section_name": "Quantitation of AbVF in the nanoconjugates using thermogravimetric analysis (TGA)",
"section_num": null
},
{
"section_content": "To compare gold nanoconjugates containing AbVF (Au-AbVF) to AbVF alone, a humanized anti-VEGF-A monoclonal antibody 2C3 (Pergerine Pharma) was co-cultured with purified CLL B primary cells.In brief, CLL B cells (1 × 10 6 ) were co-cultured with increasing concentrations of 2C3 conjugated to gold as well as 2C3 and gold (1 µg-25 µg/ml) alone as controls for 24-72 hours.Annexin/PI flow cytometry was done on those cells according to our prior procedures [3].We wish to indicate here that all the experiments reported here were repeated in triplicate. ",
"section_name": "Studying apoptosis in CLL-B cells isolated from patients",
"section_num": null
},
{
"section_content": "To detect alterations of apoptotic proteins and confirm apoptosis we used immunoblot analysis.Primary CLL B cells were cultured in six-well tissue culture dishes and treated with either vehicle (control) or Au alone, AbVF, Au-AbVF for 24 h.The treated cells were then washed three times with calcium, magnesium-free Dulbecco's phosphate-buffered saline (PBS) and solubilized in alkylation buffer (6 M guanidine hydrochloride, 250 mM Tris-HCl, pH 7.4 at 21.1°C, and 10 mM EDTA supplemented immediately before use with 150 mM 2-mecaptoethanol and 1 mM phenylmethylsulfonyl fluoride).Samples were then dialyzed into 4 M urea and then 0.1% SDS.Protein extracts were then separated on 5-15% SDS-PAGE acrylamide gels and transferred to nitrocellulose membrane and blocked with 10% Tris-saline milk (TSM).Relevant primary antibodies were diluted in the 10% TSM milk and incubated overnight on the shaker at room temperature.After three extensive washing with 1 × PBS containing 0.05% Tween 20 for 15 min, the blot was then incubated with horseradish peroxidase-conjugated secondary antibody (KPL Inc., Gaithersburg, MD, USA) diluted 1:8000 in 10% TSM for 1 h at room temperature. After washing with 1× PBS containing 0.05% Tween 20 for another 30 min, bound antibodies were detected by ECL chemiluminescence (Amersham Pharmacia, Piscataway, NJ, USA). ",
"section_name": "Immunoblotting analysis",
"section_num": null
},
{
"section_content": "TEM sample preparation involving cells, however, was performed by treating cells with gold nanoparticles and gold nanoconjugates for 1 h with under serum-free conditions.After the incubation, CLL-B cells were centrifuged initially at 2500 rpm for 6 min.The resultant cell pellets were then washed thrice with PBS, and fixed in Trump's fixative (1% glutaraldehyde and 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2).Both cell types were then rinsed for 30 min in 3 changes of 0.1 M phosphate buffer, pH 7.2, followed by a 1 hr postfix in phosphate-buffered 1% OsO 4 .After rinsing in 3 changes of distilled water for 30 min, the tissue was en bloc stained with 2% uranyl acetate for 30 min at 60°C.The cell was then rinsed in three changes of distilled water, dehydrated in progressively higher concentrations of ethanol and 100% propylene oxide, and embedded in Spurr's resin.Thin (90 nm) sections were cut on a Reichert Ultracut E ultramicrotome, placed on 200 mesh copper grids, and stained with lead citrate.Micrographs were taken on a TECNAI 12 operating at 120 KV. ",
"section_name": "Transmission electron microscopy",
"section_num": null
}
] |
[
{
"section_content": "This work is supported by CLL-Global Foundation grant to PM (CLL-2-2A3450) and a developmental grant from Hem-malignancy program at Mayo Clinic (RAF-20P) and partially supported by HL72178-05 to DM.We thank S. King of Pergerine Pharma for kindly providing the VEGF antibody.The authors are also grateful to J. Charlesworth of EM core facility at Mayo Clinic for the TEM analysis. ",
"section_name": "Acknowledgements",
"section_num": null
}
] |
10.21203/rs.3.rs-2904669/v1
|
Assessment and Prognostic Significance of a Serum Cytokine Panel in Diffuse Large B-cell Lymphoma
|
<jats:title>Abstract</jats:title><jats:p><jats:bold>Objective</jats:bold>To assess the contents of circulating cytokines in patients with diffuse large B-cell lymphoma (DLBCL), and to examine their relationship with clinicopathological manifestations and prognosis.<jats:bold>Method</jats:bold>We recruited 72 DLBCL patients, 11 chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) patients, and 56 healthy controls from our hospital between the period of January 2017 and January 2020, and measured 7 serum cytokine contents using Beckman Navios flow cytometry. The cytokine level was compared between DLBCL patients and healthy controls using one way ANOVA. Two-sided Spearman test was employed for relationship evaluation between circulating cytokine levels and clinicopathological characteristics, IPI score, and short-term treatment response within DLBCL patients. The inter-group comparison of cytokine levels employed the Mann Whitney test. The support vector machine (SVM) was utilized for the cytokine evaluation-based prediction of DLBCL patient short-term treatment response. Lastly, survival curves were used to assess correlation between the aforementioned cytokines and overall survival.<jats:bold>Result</jats:bold>The IL-6, IL-10, and IFN-γcontents were markedly enhanced among DLBCL patients, as opposed to healthy controls (<jats:italic>P</jats:italic> < 0.05). Patients with enhanced circulating LDH expressed elevated IL-10 level (P < 0.05), and patients with augmented CRP expressed upregulated IL-6 and IL-10 levels, and the rise in IL-6 levels was positively associated with serum CRP (<jats:italic>P</jats:italic> = 0.00,<jats:italic>r</jats:italic> = 0.66). Additionally, the international prognostic index (IPI) risk stratification of DLBCL patients was strongly associated with the circulating IL-6 and IL-10 contents. Enhanced IL-6, IL-10, and TNF-α levels often produced worse short-term treatment efficacies (<jats:italic>P</jats:italic> < 0.05). Moreover, the accuracy of short-term treatment response prediction model of DLBCL patients, obtained using SVM, was 81.63%. Using long-term follow-up, we further revealed that the DLBCL patients who expired within one year exhibited enhanced circulating IL-6 and IL-10 levels, compared to patients who survived, however, the IL-17 level was drastically reduced (<jats:italic>P</jats:italic> < 0.05). Despite the aforementioned evidences, we observed no marked association between the specified cytokines and overall survival (OS).<jats:bold>Conclusion</jats:bold>The IL-6, IL-10, IL-17, TNF-α, and IFN-γ contents can potentially serve as biological indicators of DLBCL tumor immune status, and a combined application with the IPI score can be a robust indicator for DLBCL patient prognosis. Our findings provide novel ideas for the clinical treatment of DLBCL patients.</jats:p>
|
[
{
"section_content": "Diffuse large B-cell lymphoma (DLBCL) is a mature B-cell malignancy that is frequently found among adults with non-Hodgkin's lymphoma (NHL) [1].In China, DLBCL accounts for 40.1% of all NHL incidences, and it has garnered increasing attention due to its enhanced morbidity and lethality.At present, DLBCL pathogenesis is unclear, and based on a few reports, DLBCL is closely associated with in ammation-triggered immune dysfunction, a condition mediated by cytokines [2,3].R-CHOP (rituximab, cyclophosphamide, adriamycin, vincristine, and prednisone) is the current rst-line treatment for DLBCL, and it produces satisfactory remission rates in most patients.However, there is a considerable proportion of patients who are either insensitive to R-CHOP or relapse early [4,5].Owing to the high heterogeneity of DLBCL patient prognosis, it is extremely crucial to establish an appropriate patient prognostic strati cation method.The international prognostic index (IPI) score, a current gold standard for DLBCL patient prognostic assessment, does not accurately re ect the role of immune escape in disease progression.Moreover, the IPI accuracy is remarkably low with standard treatment, even in combination with rituximab (R) [6].Thus, it is essential to identify novel biomarkers to enhance the IPI scoring system.Foreign investigations revealed that the expression pro les of certain cytokines are strongly correlated with the onset, severity, and prognosis of DLBCL [7][8][9].At present, there are limited relevant studies in China.In view of the differences observed between domestic and foreign environments, patient pathogenic factors, and high DLBCL heterogeneity, herein, we examined the cytokine expression pro les of DLBCL patients in China using ow cytometry.Our ndings will provide new ideas for the prognostic analysis and clinical treatment of DLBCL worldwide. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "In all, we recruited 111 patients with de nite B-NHL, con rmed via pathological biopsy, from our hospital between January 2017 and January 2020, and our diagnostic criteria were in accordance with the reported literature.Among the selected participants were 11 CLL/SLL patients (6 males and 5 females, age 55-85 years, median age 72 years); 72 primary DLBCL patients (46 males, 26 females, age 36-91 years, median age 65 years); and 21 DLBCL patients in remission after treatment ( age 36-74 years, median age 61 years).Among patients who were excluded from the study were those with autoimmune diseases, severe infections, secondary tumors, and other blood disorders.In addition, we employed 56 healthy volunteers as the control cohort (34 males and 22 females, age 24-78 years, median age 55 years).The gender and age of all four subject cohorts were comparable to one another (P = 0.461, 0.114).The nal follow-up was conducted in January 2021; the minimum follow-up period was 12 months; and the follow-up was terminated upon patient expiration during follow-up. ",
"section_name": "General information",
"section_num": null
},
{
"section_content": "We collected general clinical information from all DLBCL patients, namely, lactate dehydrogenase (LDH), C-reactive protein (CRP), ultrasonography, imaging, such as, radiographs, tomography (CT) or PET-CT, bone marrow aspiration, and biopsy.Patients also received an IPI score based on 5 factors including age, behavioral status, Ann Arbor stage, LDH expression level, and number of invaded sites of extra nodal lesions. Among the 72 DLBCL patients, three were automatically discharged without treatment, and 69 were treated with chemotherapy, according to the R-CHOP regimen over a 21-day treatment period.All patients were treated for 3 courses, prior to the e cacy evaluation, based on the NCCN Clinical Practice Guidelines in Oncology: B-Cell Lymphomas .The complete and partial remission patients made up the effectively treated cohort of 21 cases, and the disease progression and non-remission patients were classi ed under the ineffectively treated cohort of 28 cases. In all, 39 DLBC patients had available long-term follow-up data, and the remaining were lost to follow-up.In addition, 16 patients expired during a span of 1-year (follow-up period), and 23 patients survived during the period ≥ 1 year.These patients were categorized into the deceased and survived cohorts. ",
"section_name": "Sub-cohort of DLBCL patients",
"section_num": null
},
{
"section_content": "We employed a Beckman Navios ow cytometer and the human Beckman Th1/Th2/Th17 CBA cytokine kit (as per kit directions) to assess the serum concentrations of 7 speci ed cytokines among the untreated patients and healthy controls in each group prior to treatment.We collected 5 ml of fasting venous blood (arm) from all subjects, incubated at room temperature for 2 h, then separated the serum via a 5-min centrifugation at 1000g, prior to storage at 4℃.The interleukin (IL)-2, IL-4, IL-6, IL-10, IL-17, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ measurement was completed within 24 hours. ",
"section_name": "Cytokine assays",
"section_num": null
},
{
"section_content": "The SPSS 20.0 statistical analysis software was employed for all data analyses.Multi-group counting data was assessed using one-way ANOVA.Comparisons in cytokine expressions were done with the Mann-Whitney test.The Kruskal Wallis test was employed for the comparison of cytokine pro le differences between the aforementioned two patient cohorts.Lastly, we performed correlation analysis based on cytokines and different CRP levels and IPI scores using the two-sided Spearman test.Signi cance was considered at P < 0.05. ",
"section_name": "Statistical methods",
"section_num": null
},
{
"section_content": "Support vector machine (SVM) was utilized for the DLBCL patient prognosis prediction model, and it was generated via combination of the analytical results of cytokines, as determined by ow cytometry. ",
"section_name": "Generation of the prediction model",
"section_num": null
},
{
"section_content": "Expression pro les of 7 cytokines among the primary DLBCL patients, primary CLL patients, and normal control volunteers The IL-6, IL-10 (P < 0.01), and IFN-γ (P < 0.05) contents were markedly enhanced among primary DLBCL patients versus controls (Table 1).Meanwhile, the TNF-α levels exhibited a slight increase in primary DLBCL patients, but, it did not reach signi cance.We also observed no changes in the IL-2, IL-4, and IL-17 expressions between the two cohort (P > 0.05, Table 1, Supplementary Fig. 1).Moreover, unlike comparison with the primary DLBCL patients, we observed no statistical differences in cytokine levels between the primary CLL patients and healthy controls (P > 0.05, Table 1), suggesting that the serum cytokine levels are speci c to the early diagnosis of DLBCL patients. ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Apart from a rise in the serum IL-6 levels (P < 0.05), we observed no signi cant alterations in the serum cytokine expressions of 21 DLBCL patients who achieved sustained remission following standard treatment and healthy controls during the sustained remission period (P > 0.05, Table 2).This suggested an intimate relationship between cytokine expressions and disease progression among DLBCL patients. ",
"section_name": "Cytokine expression in DLBCL patients in sustained remission after treatment",
"section_num": null
},
{
"section_content": "Elevated LDH expression among DLBCL patients is closely associated with poor prognosis.Herein, we revealed that patients with enhanced serum LDH expressed considerably higher IL-6 and IL-10 contents than patients with normal LDH (Z = 2.368, 3.143, P = 0.018, 0.002), while the remaining cytokines were not signi cantly different (P > 0.05, Fig. 1).Likewise, we revealed that patients with elevated CRP exhibited markedly higher serum IL-6 and IL-10 levels, compared to patients with normal CRP (Z = 3.257, 2.159, P = 0.001, 0.031, Fig. 2A-E).Moreover, the magnitude of IL-6 elevation displayed a signi cant positive correlation with serum CRP (P = 0.00, r = 0.66, Fig. 2F).Alternately, both TNF-α and IFN-γ exhibited a signi cant trend of elevation only in patients with CRP > 50 mg/L and > 100 mg/L, respectively (P < 0.05, Supplementary Table 1).2), and there was a positive correlation between the IPI score and serum IL-6 and IL-10 levels (IL-6: P = 0.007, r = 0.380; IL-10: P = 0.002, r = 0.438, Fig. 3I).Moreover, even though the TNF-α difference between the two cohorts did not reach statistical (H = 3,474, P = 0.324), there was a substantial elevation in the cohort with IPI of 2, relative to the cohort with IPI of ≥ 3 (Fig. 3E). ",
"section_name": "Association between serum cytokine levels and clinicopathological features among DLBCL patients at rst presentation",
"section_num": null
},
{
"section_content": "The predictive model of short-term treatment response among DLBCL patients using the support vector machine (SVM) analysis of cytokines The 49 primary DLBCL patients included 21 effectively treated and 28 ineffectively treated patients.Based on our analysis, compared to the effectively treated patients, the ineffectively treated patients expressed higher serum IL-6 and IL-10 levels (P < 0.01), as well as TNF-α levels (P < 0.05, Table 3).Alternately, the IFN-γ content was not statistically different between the two cohorts, despite a strong rise among the ineffectively treated versus effectively treated patients (Fig. 4). The aforementioned cytokine data was separated into two categories, 80% for training and 20% for validation.Using the e1071 package and the random number set to 123, we performed optimization of the penalty coe cient C via tune.svm.The optimization range was between 0.005-1, optimization step was 0.005, and gamma was set to 1. Lastly, the optimal SVM model was derived as follows: the type was C-classi cation, the SVM kernel was radial, and the optimal C was 0.895.Using this optimal SVM model, the prediction test group accuracy was 78.57% (Supplementary Fig. 2), and the area under the ROC curve was 0.73 (Fig. 5). ",
"section_name": "Associations between serum cytokine pro les and IPI scores among DLBCL patients at rst presentation",
"section_num": null
},
{
"section_content": "Among the 39 DLBCL patients with long-term follow-up, 16 expired (deceased cohort) and 23 survived (survived cohort) during the 12-month follow-up.The deceased patients expressed higher serum IL-6 and IL-10 levels, whereas, the serum IL-17 content was higher among the survived patients (P < 0.05), and there were no signi cant changes in the remaining cytokines (P > 0.05, Table 4).Interestingly, based on the survival plots, IL-6 alone did not signi cantly correlate with the survival prognosis of DLBCL patients (Fig. 6). ",
"section_name": "Relationship between clinical survival and serum cytokine levels among DLBCL patients",
"section_num": null
},
{
"section_content": "DLBCL is a highly aggressive form of NHL, and it is associated with an enhanced morbidity and mortality rate.Thus, it is imperative to identify novel observational indicators that can assist in the early diagnosis, clinical treatment, and assessment of DLBCL patient prognosis.More recently, the detection of cytokines and related receptors expression levels have become an essential component of basic and clinical immunology research, as the serum contents of cytokines gain increasing importance in the elds of early diagnosis of clinical diseases, prognosis, and even in assessing the e cacies of antitumor drugs and formulating individualized treatment plans [10].Herein, we detected the cytokine expression levels of DLBCL patients using ow cytometry in order to provide novel ideas for the early diagnosis, clinical treatment and prognostic analysis of DLBCL. In recent decades, in the process of exploring new methods for the diagnosis and treatment of B-NHL, particularly DLBCL, research on IL-6, IL-10, TNF-α, and IFN-γ detection has gradually increased.IL-6 is a potent cytokine that accelerates the growth and differentiation of B lymphocytes, and it is an essential component of the lymphoma microenvironment.It also induces tumor blood vessel formation, destroys adhesion between tumor cells, and strongly prevents the anti-tumor effect of the body, thereby promoting growth, differentiation, and anti-apoptosis of tumor cells, which initiates a vicious cycle [11,12].Although IL-10 is known to possess antitumor effects mediated by CD8 + T cell responses, in the presence of CD19 + tumor cells, elevated IL-10 levels can potentially serve as a growth factor for tumorigenic B lymphocytes, and facilitate tumor cells to escape the immune system in an autocrine form [13,14].Moreover, it protects tumor cells from apoptosis via upregulation of the Bcl-2 expression, which provides an overall pro-tumor effect [15].TNF-α is among the rst cytokines released during in ammation, and it is critical for the initiation of the cytokine cascade.Elevated serum TNF-α and associated receptors (sTNF-R1 and sTNF-R2) levels are strongly associated with shorter overall survival in numerous tumors [16,17].However, the role of TNF-α in DLBCL remains unclear.IFN-γ has classical antitumor effects, but it may be tolerated or even surpassed by its pro-tumor effects in the tumor microenvironment.Some studies demonstrated that IFN-γ greatly enhances oncogenic activity via the B invasive lymphoma protein 1/ADP ribose convertase 9 (BAL1/ARTD9) in high-risk DLBCL patients [18].The role of IL-17, a characteristic Th17 cell-secreted cytokine in tumors, remains controversial.It was earlier demonstrated that in B-NHL, elevated transforming growth factor beta (TGF-β) levels inhibit Th17 cell differentiation while promoting differentiation of regulatory T cells, which produces a weakened anti-tumor mechanism, thereby enabling tumor immune escape [19]. Herein, we demonstrated signi cantly different expressions of the DLBCL and CLL cytokines, two typical representatives of aggressive and inert B-cell lymphomas.The expressions of IL-6, IL-10, and IFN-γ were signi cantly higher among DLBCL patients than in healthy controls.TNF-α was also strongly upregulated between the two cohorts, which con rmed that the correlation between aberrant cytokine expressions and DLBCL occurrence.No signi cant differences were observed in these cytokines between the CLL/SLL patients and healthy controls. Although some prior studies revealed that the serum IL-6 and IL-10 levels remain elevated in CLL/SLL patients, these are primarily concentrated within certain high-grade patients [20].In this study, we demonstrated to a certain extent that serum cytokines have limited diagnostic signi cance for the inert B-cell lymphoma represented by CLL, however, it is speci c for DLBCL.Additionally, among DLBCL patients, the aforementioned cytokines, apart from IL-6, often returned to normal levels upon continued remission of the disease.This suggested that cytokines also have a strong relationship with disease progression, thus providing an idea for the treatment of DLBCL patients. Meanwhile, elevated LDH and CRP expressions were associated with poor prognosis in DLBCL patients, and we revealed via grouping that patients with enhanced serum CRP or LDH levels expressed higher IL-6 and IL-10 contents, and that the elevation of these factors was positively associated with serum CRP, which was consistent with the reports of Nacinovic et al [21].Furthermore, there was a strong correlation between the IPI risk strati cation and circulating IL-6 and IL-10 levels in DLBCL patients.The IL-10 was signi cantly higher in low-risk IPI patients, relative to the high-risk IPI patients, and the difference was greater with elevated scores.Our e cacy observations revealed that elevated IL-6, IL-10, and TNF-α levels often predicted worse treatment effectiveness, which was consistent with the ndings of Dlouhy et al [7].We further tested cytokines using the SVM technique, and the accuracy of our optimal SVM model to predict short-term treatment e cacies among DLBCL patients was 81.63%. To a certain extent, this con rmed that cytokines are an important indicator of DLBCL treatment response. Using long-term follow-up, we demonstrated that DLBCL patients who expired within one clinical year expressed enhanced serum IL-6 and IL-10 levels, as well as strongly diminished IL-17 levels, compared to the patients who survived.In contrast, using survival curves, we did not observe a signi cant association between IL-6 levels and overall survival of DLBCL patients.This suggested, to some extent, that serum cytokines have some limitations in indicating long-term prognosis of DLBCL patients. In conclusion, the IL-6, IL-10, IL-17, TNF-α and IFN-γ contents can serve as prognostic indicators for the assessment of tumor immune status in DLBCL.Moreover, in combination with the IPI score, they can be important indicators of DLBCL prognosis, and may also provide a basis for the precise treatment and direction of novel and e cacious targeted therapies.Tables ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "Not applicable. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This study was supported by the Foundation of Science Technology Department of Zhejiang Province (No. LGF22H080012), Zhejiang Provincial Medical Technology Plan Project (No. 2022KY505, No. 2020KY052). ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "Not applicable. ",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "Authors' contributions SX, LZ and LW collected and analyzed the data, drew fgures and tables, and contributed in writing the manuscript.SW, LZ and SX performed the statistical analysis; XT procured the funding for this study.XT, WN, SW participated in the design of the study, gave administrative or logistical support for this study, and reviewed drafts of the paper.All the authors agreed with the conclusions of this review and approved the nal manuscript. This study was authorized by the Ethics Committee of the Zhejiang Provincial People's Hospital (2021QT150). Not applicable. The authors declare that they have no competing interests. The ROC curve of circulating cytokine SVM prediction model for the short-term treatment response of DLBCL patients. The survival curve analysis of the individual serum factor IL-6 in DLBCL patients. This is a list of supplementary les associated with this preprint.Click to download. supplementary gureandtable.zip ",
"section_name": "Declarations",
"section_num": null
},
{
"section_content": "Authors' contributions SX, LZ and LW collected and analyzed the data, drew fgures and tables, and contributed in writing the manuscript.SW, LZ and SX performed the statistical analysis; XT procured the funding for this study.XT, WN, SW participated in the design of the study, gave administrative or logistical support for this study, and reviewed drafts of the paper.All the authors agreed with the conclusions of this review and approved the nal manuscript. ",
"section_name": "Declarations",
"section_num": null
},
{
"section_content": "This study was authorized by the Ethics Committee of the Zhejiang Provincial People's Hospital (2021QT150). ",
"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": "The ROC curve of circulating cytokine SVM prediction model for the short-term treatment response of DLBCL patients. The survival curve analysis of the individual serum factor IL-6 in DLBCL patients. ",
"section_name": "Groups",
"section_num": null
},
{
"section_content": "This is a list of supplementary les associated with this preprint.Click to download. supplementary gureandtable.zip ",
"section_name": "Supplementary Files",
"section_num": null
}
] |
10.1186/1755-8166-6-5
|
Atypical rearrangement involving 3′-IGH@ and a breakpoint at least 400 Kb upstream of an intact MYC in a CLL patient with an apparently balanced t(8;14)(q24.1;q32) and negative MYC expression
|
<jats:title>Abstract</jats:title> <jats:p>The t(8;14)(q24.1;q32), the cytogenetic hallmark of Burkitt’s lymphoma, is also found, but rarely, in cases of chronic lymphocytic leukemia (CLL). Such translocation typically results in a <jats:italic>MYC</jats:italic>-<jats:italic>IGH@</jats:italic> fusion subsequently deregulating and overexpressing <jats:italic>MYC</jats:italic> on der 14q32. In CLL, atypical rearrangements resulting in its gain or loss, within or outside of <jats:italic>IGH@</jats:italic> or <jats:italic>MYC</jats:italic> locus, have been reported, but their clinical significance remains uncertain. Herein, we report a 67 year-old male with complex cytogenetic findings of apparently balanced t(8;14) and unreported complex rearrangements of <jats:italic>IGH@</jats:italic> and <jats:italic>MYC</jats:italic> loci. His clinical, morphological and immunophenotypic features were consistent with the diagnosis of CLL.</jats:p> <jats:p>Interphase FISH studies revealed deletions of 11q22.3 and 13q14.3, and an extra copy of <jats:italic>IGH@,</jats:italic> indicative of rearrangement. Karyotype analysis showed an apparently balanced t(8;14)(q24.1;q32). Sequential GPG-metaphase FISH studies revealed abnormal signal patterns: rearrangement of IGH break apart probe with the 5’-IGH@ on derivative 8q24.1 and the 3’-IGH@ retained on der 14q; absence of MYC break apart-specific signal on der 8q; and, the presence of unsplit 5’-<jats:italic>MYC</jats:italic>-3’ break apart probe signals on der 14q. The breakpoint on 8q24.1 was found to be at least 400 Kb upstream of 5’ of <jats:italic>MYC</jats:italic>. In addition, FISH studies revealed two abnormal clones; one with 13q14.3 deletion, and the other, with concurrent 11q deletion and atypical rearrangements. Chromosome microarray analysis (CMA) detected a 7.1 Mb deletion on 11q22.3-q23.3 including <jats:italic>ATM</jats:italic>, a finding consistent with FISH results. While no significant copy number gain or loss observed on chromosomes 8, 12 and 13, a 455 Kb microdeletion of uncertain clinical significance was detected on 14q32.33. Immunohistochemistry showed co-expression of CD19, CD5, and CD23, positive ZAP-70 expression and absence of <jats:italic>MYC</jats:italic> expression. Overall findings reveal an apparently balanced t(8;14) and atypical complex rearrangements involving 3’-<jats:italic>IGH@</jats:italic> and a breakpoint at least 400 Kb upstream of <jats:italic>MYC</jats:italic>, resulting in the relocation of the intact 5’-<jats:italic>MYC</jats:italic>-3’ from der 8q, and apposition to 3’-<jats:italic>IGH@</jats:italic> at der 14q. This case report provides unique and additional cytogenetic data that may be of clinical significance in such a rare finding in CLL. It also highlights the utility of conventional and sequential metaphase FISH in understanding complex chromosome anomalies and their association with other clinical findings in patients with CLL. To the best of our knowledge, this is the first CLL reported case with such an atypical rearrangement in a patient with a negative <jats:italic>MYC</jats:italic> expression.</jats:p>
|
[
{
"section_content": "Chronic lymphocyctic leukemia (CLL) is the most common leukemia in the elderly with clinical presentation of lymphocytosis, bone marrow involvement, lymphadenopathy, hepatosplenomegaly, complex cytogenetics and heterogeneous clinical course [1].Immunophenotypically, aberrant expression of CD5, CD20, CD22, CD23, CD38, CD43 and CD79 is diagnostic or prognostic of B-cells in CLL [2].Common cytogenetic anomalies include deletion of 13q14.3(most frequent) and/or 13q34, deletion of 11q, deletion of 17p, trisomy 12 and IGH@ rearrangement [3]. While t(8;14)(q24.1;q32), the cytogenetic hallmark of Burkitt's lymphoma, is a primary genetic event found in about 70-80% of cases, it is usually a rare secondary anomaly in other B-cell disorders including CLL (about 0.2% to <1%) [4][5][6][7][8], lymphoblastic leukemia, DLBCL, and in other lymphoma transforming into a more aggressive disease [9].In the latter, t(8;14) usually confers favorable prognosis, while a more aggressive phenotype and poor outcome are manifested when it is a part of a complex chromosome complement [5,10]. In a typical t(8;14)(q24;q32) translocation, the MYC at 8q24.1 locus is spatiotemporally juxtaposed with the 3'-IGH@ locus on derivative 14q32 [11][12][13][14][15].The IGH transcription factory, about 2.5 Mb in size [12], localizes the regulatory elements for MYC deregulation and variable regions that promote translocation [13].The IGH@ locus, is a hotspot for recombination and mutation of immunoglobulin genes during B-cell maturation, processes that usually promote translocations with oncogenic potential [11].Whereas the breakpoint on chromosome 14 is within the IGH@ locus, usually located within the μ-gene, either within or adjacent to the variable (V), joining (J), diversity (D, or switch (S) regions, but other heavy-chain regions are occasionally involved [9].While about 80% of translocations in Burkitt's lymphoma is typical and involve MYC and IGH@ (IG heavy chain) [16], others are involved in variant partnership with other IG chain loci; kappa light chain (IGK) at 2p12, or lambda light chain (IGL) at 22q11.2 [16][17][18].MYC is also involved with IGH in DLBCL [18], TCR alpha/delta in T-acute lymphoblastic leukemia/lymphoma, and IG kappa and lambda chains in plasma cell myeloma [18,19]. MYC is a proto-oncogene that encodes for a transcription factor that regulates cell cycle progression, growth, differentiation, apoptosis, survival and biosynthesis [4,6,20].It activates or represses transcription factories of other genes (about 10%), transcription factors, and chromatin modifying and remodeling complexes [20].Rearrangements involving MYC drive cells into lymphomagenesis often through its deregulation and overexpression [5,11,12,21,22].The oncogenic potential of MYC rearrangements is implicated not only in the initiation of lymphomagenesis but also in its transformation and progression of low-grade lymphomas into a more advanced disease and an unfavorable outcome [5,17,18,[21][22][23].These findings suggest that the level of deregulated MYC expression of different stages of aberrant cellular maturation and differentiation may influence the neoplastic phenotype [9].At 8q24.1 locus, translocation breakpoints are located within or surrounding the MYC: regulatory region within MYC, from exon 1 to intron 1, (Class I and most common); transcription factor binding-site at or adjacent to 5'-MYC (Class II); and long-range regions up to 100-300 Kb or more upstream from an intact 5'-MYC-3' (Class III) [15,16,20].It has been suggested that aberrant MYC expression is influenced by breakpoint location, mutation within the translocated region, deletion of regulatory elements, or transcription at cryptic sites other than the usual P1 or P2 initiation start site (promoter shift) [15,20,24].Increased transcriptional activity is observed in breakpoints within exon 1 and intron 1 (Class I) than when it occurs within the most common breakpoint, 5' from MYC exon 1 (Class II) [15].Longrange cis-acting enhancers regulate MYC expression through chromatin looping bringing the enhancers in close proximity to MYC [25,26], or through increased distal enhancer activity utilizing preexisting loops [27].Multiple genetic variants and SNPs, located in 1.5 Mb \"gene desert\" regions 1, 2 and 3, up to 600 Kb upstream of MYC, are associated with increased susceptibility to prostate, colorectal, bladder, breast cancer, or chronic lymphocytic leukemia [26][27][28].Although reporter expression studies revealed that long-range enhancers and other regulatory elements regulate MYC transcription, the clinical significance of MYC rearrangements upstream of MYC remain unclear and a subject of burgeoning field of investigation [4]. To date, there are only very few reported cases of CLL with apparently balanced t(8;14) and atypical rearrangements [6,8], none of which exhibits abnormal FISH signal patterns similar to what we detected in our patient.These abnormal patterns include: cryptic deletion on 8q24.1 including MYC [6,8], gain of an extra copy of MYC (+MYC) [4,5,29], or deletion of IGH@, usually 5' [3,4,6,30,31].The prognosis for these cases is also variable, from indolent to transformed into a more aggressive course. Here we report a CLL case with complex cytogenetic findings of deletions of 11q and 13q, in addition to the apparently balanced t (8;14).We also present an undocumented atypical complex rearrangements involving 3'-IGH@ and at least 400 Kb upstream of 5'-MYC, unreported complex atypical rearrangements of IGH@ and MYC loci that did not result in IGH-MYC fusion and no subsequent MYC expression. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "Our patient is a 67 year-old Hispanic male with a medical past history of an end-stage kidney disease of uncertain etiology.His white blood cell count (WBC) was elevated and measured at 23.15 × 10 3 per μL.Peripheral blood smear showed marked lymphocytosis with numerous atypical lymphoid cells including prolymphocytes, smudge cells, normocytic normochromic anemia and thrombocytopenia.The lymphoid-gated population constituted 87% of total cells, and consisted of 2% T cells, 70% B cells, and <2% NK cells (Figure 1A).Flow cytometry showed co-expression of B-cell antigen (CD19) with CD5, CD23, CD20, and ZAP70 expression.These results were suggestive of CLL (Figure 1B). ",
"section_name": "Clinical report",
"section_num": null
},
{
"section_content": "A complete chromosome analysis was not possible due to low mitotic index.G-banded karyotype analysis of available metaphase cells revealed an abnormal male karyotype with an apparently balanced t(8;14)(q24.1;q32)seen in 50% (6/12) of total cells examined (Figure 2). Interphase FISH studies did not reveal IGH@-CCND1 rearrangement, but instead, an extra copy of IGH@-specific signal in 30.3% (91/300) of nuclei examined (data not shown).In addition, deletions of the 13q14.3(D13S319) (Figure 3A) and 11q22.3(ATM) (Figure 3B) in 8% (24/300) and 78% (294/300) of cells were also observed, respectively.Neither deletion of 17p13.1 (TP53) nor trisomy 12 was detected (data not shown).Sequential GPG-metaphase FISH studies were performed on the same chromosome metaphase spread to determine the clonality of the structural abnormalities seen in our patient.Results showed that two different clones exist in the peripheral blood of our patient: one with deletion 13q14.3(seen only in interphase nuclei in our study), and another with concurrent deletion 11q (Figure 3C) and t(8;14) (Figure 4D). Further sequential FISH studies on 10 metaphase nuclei using the IGH@ break apart probe showed splitting or rearrangement (1Y1G1R), with the 5'-IGH@ (green) translocated on chromosome 8q24.1 and the 3'-IGH@ (red) retained on 14q (Figure 4A,B) in all cells examined.The IGH@-MYC fusion (Figure 4C) and MYC break apart (Figure 4D) probes revealed atypical abnormal signal patterns in all 10 cells examined on derivative 8q24.1:one green (5'-IGH@) and no red (deletion at least 400 Kb upstream of 5'-MYC-3'); and, on derivative 14q32: one yellow (relocation of 5'-MYC-3' and its flanking regions adjacent to 3'-IGH@).The estimated location of the translocation breakpoint upstream of 5'-MYC was determined by in silico mapping (Figure 5) by determining the base pair coordinates in the UCSC Genome Browser (hg19) of the STS markers mapped upstream of 5'-MYC-3' (Abbott Vysis FISH probes website).We based our calculations on the Spectrum Orange of the MYC break apart probe, the farthest probe from 5' of MYC (as compared to the MYC probe in the IGH-MYC fusion probe).The estimated distance of the translocation breakpoint (STS marker WI-1302) from 5'of MYC is at least 400 Kb (bp 128,354,420-128,747,680).This interval includes two RefSeq genes: POU5F1B (POU class 5 homeobox 1B), an intronless gene that encodes for a transcription factor (1.6 Mb; bp 128,427,857-128,429,441) and a gene with no known function, LOC727677 (38.8 Kb; bp 128,455,595-128,494,384).It also includes the SNPs implicated in several cancer types, rs1447295 (Region 1), rs16901979 (Region 2) and rs6983267 (Region 3) [26] and CLL SNP rs2456449 [28]. SNP CMA refined the 11q22.copy number loss at chromosome 11q22.3-q23.3,arr 11q22.3q23.3(107,888,769-115,016,307)x1(data not shown).It deleted 62 RefSeq genes including ATM (ataxia telangiectasia mutated), a gene that encodes for a cell cycle checkpoint phosphorylating kinase that functions for regulating proteins for tumor suppression, checkpoint, DNA repair and maintenance of genome stability [32].In addition, a 455 Kb heterozygous copy number loss on 14q32.33 was also detected; arr 14q32.33(106,530,533-106,985,955)x1,deleting two gene fragments or non-protein coding genes of no known function, LINC00226 and LINC00221 (data not shown) [32].CMA did not detect a microdeletion within or surrounding the MYC locus despite its removal from der 8q24.1 locus.This suggests that there was no net gain or loss despite the unbalanced rearrangements detected by FISH.In a lesser extent, a 61 Kb gain on 8q24.12 was detected, but found to be unreportable with further in silico investigations.There were no clinically relevant gains or losses detected on chromosomes 12, 13 and 17. According to the ISCN [33], the overall findings from karyotype, FISH and CMA can be described as: 46,XY,t(8;14)(q24.1;q32).ishder(8)t(8;14)(q24.1;q32)del(8)(q24.1q24.1)(MYC-,5'IGH@+),der(14 As mentioned above, deletion 13q14.3 and deletion 11q22.3 with t(8;14), detected by interphase and sequential metaphase FISH studies, are found as two different abnormal clones, indicative of mosaicism.CMA failed to detect gains or losses on 13q, since it only accounts for 8% of the total cell population, a number way below the detection limit (10-30%) of either SNP or BAC microarrays [30]. Immunohistochemistry studies using specific MYC antibodies did not detect any staining in our patient's sample, suggestive of absence of MYC activation (Figure 6A).A strong positive staining for MYC was detected for the positive control sample (Figure 6B). ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Our patient's clinical, morphological and immunophenotypic features are consistent with the diagnosis of CLL.Although complex cytogenetic findings including t(8;14) usually confers poor prognosis in CLL, a consistent genotype and phenotype correlation remains an unresolved issue.Our patient's case exhibits an unreported rearrangement involving IGH@ and MYC loci with absence of MYC expression. In our patient, the FISH signal patterns detected are unique from those previously reported in CLL cases with atypical rearrangements and an apparently balanced t (8;14).These include a cryptic deletion on 8q24.1 including MYC [6,8], gain of an extra copy of MYC (+MYC) [4,5,29], or deletion of IGH@, usually 5' [3,4,6,30,31].Although a deletion of the MYC-specific signal on der 8q24.1 locus was also observed in our patient using IGH-MYC fusion probe (1Y2G1R), it is not identical to the reported deletion by Reddy et al. in 2006 [6,8].The deletion reported on here did not show splitting of signals and no concomitant deletion of a 1.6 Mb segment including the MYC locus.Instead, it showed two unsplit MYC probes (yellow) on the normal chromosome 8 and on der 14q32.We interpreted these findings as an atypical rearrangement never reported elsewhere, with the 5'-MYC-3' removed from the 8q24.1 locus at a breakpoint at least 400 Kb upstream of its 5' region.We also showed that this deleted region is relocated to the 14q32 locus and apposed to the 3'-IGH@ locus.Neither gain of MYC nor deletion of the 5'-IGH@ locus was observed by FISH or CMA in our case.We have exhaustively searched the available literature and did not find any cases similar to the signal patterns reported on here. To the best of our knowledge, expression levels of MYC and its correlation to disease progression have not been established in CLL with t(8;14), with or without MYC translocations [4,6,7].MYC expression is generally at low levels in CLL [23], and similar in groups with either bad or good prognosis Increased expression even without MYC rearrangement has also been described in CLL with malignant Richter transformation and other higher risk cases for CLL progression [10].Although, high levels of MYC are expressed as a result of the t(8;14) and its variant translocations in Burkitt's lymphoma and in some other B-cell malignancies including DLBCL and plasma cell myeloma, these translocations may not necessarily lead to increased expression of MYC in CLL [4,6,7].These variable findings of MYC expression are most likely dependent on specific disruptions of regulatory regions, or characteristic genomic translocation breakpoints either at the MYC or IGH@ locus.The typical MYC-IGH fusion at der 14q32 expresses the MYC -deregulatting product, while the reciprocal IGH-MYC fusion at 8q24.1 locus is transcriptionally silent [14,35].Despite the typical juxtaposition, overall MYC expression in some CLL cases remains within the normal range [20], or overexpressed through processes other than translocations [9].It has been reported that the location of the genomic breakpoint influences MYC expression, with highest level when involving Class I breakpoints [15,24].The absence of Myc expression in our patient is most likely due to the atypical MYC-IGH fusion on der 14q32, with a Class III breakpoint (at least 400 Kb upstream of MYC) [16]. The previously reported \"gene desert\" region upstream of MYC extends up to about 629 Kb [26] and includes genes and SNPs.Genome-wide association studies (GWAS) have shown the gene POU5F1B and several genetic variants or SNPs (Regions R1, R2, R3) (Figure 5) that are risk factors for various cancers including CLL exist in this region [26][27][28][36][37][38].The strongest evidence for risk or genetic susceptibility in CLL or monoclonal B-cell lymphocytosis is rs2456449 (8q24.21)[28,38].In our patient, the breakpoint that we suggested (at least 400 Kb) is within this interval and includes POU5F1B, and SNPs R1 and R2.POU5F1B is one of the two RefSeq genes within the breakpoint on 8q24.1 and 5'-MYC, is the most adjacent.Although it is not yet well studied, few reports described it as a pseudogene or a gene that encodes for a weak transcription factor that may play a critical role in stem cell pluripotency, eye development and carcinogenesis [36,37,39].At the present time, there are no reports of a specific fusion involving 5'-POU5F1B The IGH@ break apart probe reveals splitting of signals (1Y1G1R) indicative of rearrangement, with 5'-IGH@ relocated to der 8q and 3'-IGH@ retained on der 14q.C: The IGH-MYC fusion probe shows 1Y2G1R2A, fusion of MYC-IGH der 14q32 (yellow), and deletion of MYC on der 8q24.1 (green, no red).It also shows normal signal patterns for the other chromosome 8 (aqua for centromere and red for MYC), and chromosome 14 (green).D: The MYC break apart probe detects 2Y signals, one on normal chromosome 8 and the other is removed from der 8q24.1 and relocated to der 14q32.These findings suggest that there is neither splitting of signals, or a deletion in between the red and green signals. and 3'-regulatory region of [email protected] is possible that the breakpoint in our patient is further upstream, however, the paucity of available cells made it impossible for further characterization.In Figure 5, we extended the suggested breakpoint further upstream, from ~400 Kb to ~600 Kb, to include the farthest reported cancer-associated SNP (Region 2: rs16901979) and CLL SNP (rs2456449).To date, the genotype phenotype correlation underlying these associations remains unclear.However, it has been suggested by reported expression studies that MYC expression is influenced by such SNPs variants by altering its transcription regulation and amplification [40].Despite such plethora of reports, replication of these findings and elucidation of its physiologic function and clinical significance remain an area of thorough investigation.Further in vivo and in vitro functional studies are needed to show consistent association of risk allele status and MYC expression levels. On the other hand, transcription at the IGH@ locus is controlled by enhancers elements spread out as wide as 2.5 Mb of the locus [12], and it contains regulatory elements necessary not only for MYC activation but also the promotion of translocation [13].CMA detected a 455 Kb copy number loss on chromosome band 14q32.2,not detected by FISH since the probe used was outside of this region.It is still a possibility, that the deletion in our patient may have removed some of the regulatory elements within this interval somehow affecting the regulation of Myc expression.No regulatory elements or high conservation data was seen in the UCSC Genome Browser.This microdeletion has been reported in other CMA studies of CLL patients using BAC-based array CGH, with some of the cases exhibiting the same findings as ours, i.e. with no IGH@ deletion by FISH [30].It is still unclear whether this microdeletion is a The farthest probe upstream of 5' of MYC is the Spectrum Orange of the MYC break apart probe (upper panel) (adopted from Abbott Vysis website for FISH probes).We plotted the base coordinates for WI-1203 STS marker and the 5' of MYC (see inlet) into the UCSC Genome Browser (hg19) and determine the distance, RefSeq genes (POUF51B and LOC727677), STS markers and SNPs within this interval.The translocation breakpoint (with lightning icon) is centromeric of WI-1203 and about ~400 Kb upstream of 5'-MYC, a Class III breakpoint.We extended the breakpoint further upstream to show ~629 Kb region containing SNPs in different regions of the interval (R1, R2, R3 and CLL) that confer susceptibility risks for cancer [26,28].polymorphic feature of this locus and represents a region of frequent mutation and recombination, or it exhibits some susceptibility risks for CLL [3,24,31,41]. About 20% of patients with CLL show ATM deletion, an anomaly also seen in almost all cancer, and is usually associated with an adverse outcome [1,4,31].The collaboration of ATM and MYC in normal cell proliferation via an ATM-dependent pathway is well established.When deleted, ATM loses its protective checkpoint function leading to MYC-induced oncogenesis [4,42].This indicates that MYC alone is not capable of transforming lymphoid cells into neoplasia [4].The ATM deletion and removal and relocation of MYC observed in our patient may explain the lymphomagenesis, but not necessarily the absence of Myc expression. Given the limitations of this case report, we suggest that comprehensive retrospective studies in CLL patients should be performed to characterize the suggested ~400 Kb breakpoint and the region further upstream by sequential metaphase BAC FISH mapping since CMA does not detect the removal and relocation of an intact MYC locus.It is also possible that the absence of Myc expression is a false negative result given the specificity of immunostaining which is below 100%, and about 17% of cases may be overlooked for MYC rearrangements using this technique [43].A more accurate quantitative approach such as RT-qPCR is recommended.Since variability in MYC breakpoints could still result in similar MYC expression [44], possibly due to flexible DNA looping [43,45], reporter expression studies are needed to better understand the clinical impact and significance of long distance deregulation in in loci with atypical MYC rearrangement. This paper presents an unreported atypical rearrangement involving the IGH@ and MYC loci detected by FISH, adding to the burgeoning cytogenetic data on CLL patients with atypical t (8;14).It also highlights the Class III translocation breakpoint upstream of MYC, including the cancer and CLL-associated SNPs within the interval.This report also provides important and promising findings for further studies correlating Myc expression with a specific type of genomic translocation breakpoint or copy number variants in CLL and in other B-cell disorders.Lastly, overall findings in our report highlight the utility of karyotype analysis, interphase and sequential metaphase FISH studies, CMA, and other molecular tools in approaching the diagnosis and prognosis of CLL in a more comprehensive manner. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Conventional GPG-banded chromosomal analysis was performed on peripheral blood lymphocytes that were cultured for 48 and 72 hours with and without pokeweed mitogen stimulation, following standard cytogenetics protocols.The karyotypes were described according to the ISCN 2009 nomenclature [33]. Initial FISH studies were performed on interphase cells using CLL panel probes (Abbott Molecular, Des Plaines, Illinois) specific for centromere 12, IGH@ break apart or IGH@-CCND1 fusion, and chromosome loci 13q14.3(D13S319)/13q34, 11q22.3(ATM) and 17p13.1 (TP53).Sequential GPG-metaphase FISH studies were performed using IGH-MYC fusion (with centromere 8-specific probe), and break apart probes for the IGH@ and MYC loci. Chromosome microarray analysis was performed on DNA sample from 48-hr culture of peripheral blood lymphocytes.DNA was extracted from Carnoy's fixed pellet cells Qiagen DNA extraction kit (Valencia, CA).DNA concentration and quality was checked using Nanodrop (Life Technologies, Carlsbad, CA) and gel electrophoresis, respectively.Whole genome chromosome SNP microarray was performed to assess for imbalances (i.e. gain or losses) in the genomic DNA sample tested.The assay compared the patient's DNA to a reference set from 380 normal controls (284 HapMap and 96 Affymetrix reference), using the Genome-Wide SNP Array CytoScan HD.This array platform contains 2.6 million markers for Copy Number Variant detection (Affymetrix, Inc.), which 750,000 are genotype SNPs and 1.9 million are non-polymorphic probes, for the whole genome coverage.The analysis was performed using the chromosome analysis suite (ChAS), version CytoB-N1.2.2.271(r4615).Oligonucleotide probe information is based on the 37 build of the Human Genome (UCSC Genome Browser, http://genome.ucsc.edu/cgi-bin/hgGateway, hg19, February 2009). FFPE sections (4 μm thick) were stained for Myc using rabbit monoclonal human anti-Myc antibody (catalog #1472-1, Epitomics, Inc., Burlingame, CA, USA).Heat-induced epitope retrieval was accomplished by using ER1 for 20 min.Endogenous peroxidase was blocked using hydrogen peroxide.The slide was incubated in the primary antibody Myc for 30 min, followed by incubation in a post-primary 3,3-diaminobenzidine for 10 min, polymer 3,3-diaminobenzidine for 10 min, and chromogen 3,3-diaminobenzidine for 10 min.Subsequently, slide was incubated with post-primary alkaline phosphatase for 20 min, polymer alkaline phosphatase for 30 min, and fast red for 20 min.The nuclei were counterstained with hematoxylin and the slide was then dehydrated, cleared in xylene, and coverslipped.Appropriate positive controls were used. ",
"section_name": "Materials and methods",
"section_num": null
}
] |
[
{
"section_content": "We would like to thank the assistance of the following UCLA Cytogenetics Lab Staff: Lynn Yang for her technical assistance and her willingness to help and make this project possible.We would also like to acknowledge Ingrid Jaramillo, Gloria Lan, Pinky Bolire, Karen Park and Yun Lei and other FISH technologists in making this project possible.We also like to thank Dr. Kingshuk Das and the staff of UCLA Molecular Pathology Lab for assisting us in the DNA extraction.We are also grateful to Affymetrix for performing CMA in our patient's DNA sample. ",
"section_name": "Acknowledgement",
"section_num": null
},
{
"section_content": "The authors declare they have no competing interests. Authors' contributions IA performed metaphase studies; gathered data for karyotype, interphase and metaphase FISH studies; analyzed, interpreted and wrote the cytogenetic report; analyzed and interpreted chromosome microarray data; and did necessary revisions in the manuscript as requested by the reviewers.PHB analyzed the molecular data, correlated clinical findings, and wrote the initial draft of the paper.BS provided the flow cytometry and immunohistochemistry data.SK reviewed the microarray data and paper draft.CT analyzed and reviewed all the data and drafted the paper.All authors read and approved the final manuscript. ",
"section_name": "Competing interests",
"section_num": null
}
] |
10.1007/s11912-020-0881-4
|
Revolution of Chronic Lymphocytic Leukemia Therapy: the Chemo-Free Treatment Paradigm
|
<jats:title>Abstract</jats:title><jats:sec><jats:title>Purpose of Review</jats:title><jats:p>Over the last years, targeted anticancer therapy with small molecule inhibitors and antibodies has much replaced chemoimmunotherapy, which has been the gold standard of care for patients with chronic lymphocytic leukemia (CLL). Here we give an overview of novel targeted agents used in therapy of chronic lymphocytic leukemia, as well as efforts to overcome resistance development, focusing on approved drugs since they gained high relevance in clinical practice.</jats:p></jats:sec><jats:sec><jats:title>Recent Findings</jats:title><jats:p>Novel agents moved to the forefront as a treatment strategy of CLL due to their outstanding efficacy, almost irrespectively of the underlying genetic features. Inhibition of Bruton’s tyrosine kinase (BTK), a key molecule in the B cell receptor pathway, achieved dramatic efficacy even in poor-risk and chemo-refractory patients. Further success was accomplished with venetoclax, which specifically inhibits anti-apoptotic BCL2 and induces apoptosis of CLL cells.</jats:p></jats:sec><jats:sec><jats:title>Summary</jats:title><jats:p>Inhibition of BTK or BCL2 is very effective and induces prolongation of progression-free and overall survival. Approved combination treatments such as venetoclax or ibrutinib with obinutuzumab show high responses rates and long remission durations. However, evolution and selection of subclones with continuous treatment leads to resistance towards these novel drugs and disease relapse. Hence, comparison of sequential treatment with combinations and discontinuation of therapy are important aspects which need to be investigated.</jats:p></jats:sec>
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in adults and mainly affects the elderly [1].CLL is a B cell malignancy, where clonal CD5 + CD19 + CD23 + B cells accumulate in peripheral blood and infiltrate secondary lymphoid organs such as lymph nodes, spleen, and bone marrow [2].The disease is highly heterogeneous clinically mostly due to hypermutations of the immunoglobulin heavy-chain genes (IGHV), genomic aberrations, and recurrent gene mutations which associate with the clinical course [3,4].The mechanisms underlying CLL pathogenesis are not fully resolved.Allogenic stem cell transplantation is still the only curative therapy, although limited to a small subset of young and fit patients [5].For the last 10 years, chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab (FCR) has been the most effective treatment but its success is limited by comorbidities, age, and fitness of the patients [6][7][8].However, treatment of patients with high-risk del(17p) or TP53 mutations have shown poor outcome [9][10][11]. Recent scientific advances in understanding the biology of CLL evolved in the development of novel therapeutic agents.Small molecule inhibitors targeting key survival mechanisms revolutionized therapy and showed unparalleled effects in patients irrespective of their genetic aberrations.The novel agents led to a paradigm change in patient care from treatment with unspecific DNA damaging agents to \"targeted therapy.\" ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "The CD20 antigen is expressed on the surface of mature B cells and is one of the most successful targets in treatment of B cell malignancies.Monoclonal antibodies are widely used to deplete B cells in cancers or autoimmune diseases.The first CD20 antibody was rituximab, which has been FDA (US Food and drug administration)-approved in 1998.Several modes of action of rituximab are currently known such as complement-dependent cytotoxicity (CDC), opsonization of macrophages inducing antibodydependent cell-mediated cytotoxicity (ADCC), and direct killing by apoptosis to a lower extent [12].Additionally, CD20 receptor colocalizes with the B cell receptor participating in its activation and signaling.Despite that, the exact mode of action of rituximab still remains unclear.If rituximab was combined with fludarabine and cyclophosphamide, progression-free survival (PFS) and overall survival (OS) were significantly improved [13].In the CLL8 trial, the FCR (fludarabine-cyclophosphamide-rituximab) group had a PFS of 56.8 months compared with 32.9 months in the FC (fludarabine-cyclophosphamide) arm.Here, the median OS in the FCR arm was not reached in comparison with 86 months in the FC arm [8].IGHVmutated patients had most benefit from FCR.However, chemoimmunotherapy is less effective with negative impact on PFS and OS in patients with unmutated IGHV, mutated TP53 and del(17p), del(11q), and some gene mutations such as NOTCH1, SF3B1, and BIRC3 [3,7]. Ofatumumab is a humanized anti-CD20 monoclonal antibody which targets a different epitope than rituximab resulting in enhanced activation of CDC and similar activation of ADCC and apoptosis [14].Ofatumumab was approved as a single agent in fludarabine refractory CLL, as well as in combination with fludarabine and cyclophosphamide for refractory CLL or with chlorambucil or bendamustine for treatment-naïve patients.Good tolerability was shown in elderly patients with a median PFS of 22.4 months (ofatumumab and chlorambucil; C O M P L E M E N T-1 t r i a l ) v e r s u s 1 3 .1 m o n t h s (chlorambucil monotherapy) [15].The COMPLEMENT-2 trial demonstrated an increased PFS of relapsed patients from 18.8 months (FC) to 28.9 months (FCO) when adding ofatumumab to the FC treatment [16].Similar to treatment with rituximab, patients with NOTCH1 mutations benefited less.In spite of everything, the use of ofatumumab is suggested in earlier course of disease, since a phase IV study in heavily pretreated patients demonstrated limited efficacy and low numbers of responses [17]. On the contrary, obinutuzumab (GA101) is a recombinant type II anti-CD20 and immunoglobulin G1 Fc-optimized monoclonal antibody, which induces CDC and direct cell death upon binding to CD20 depending on actin reorganization and lysosome involvement [18].A successful phase I trial showed a response rate of 62% demonstrating activity of obinutuzumab in heavily pretreated patients [19].In the phase III CLL11 study, obinutuzumab was combined with chlorambucil (clb) and compared with rituximab-chlorambucil and chlorambucil monotherapy.Obinutuzumab-clb was superior to rituximab-clb and clb-monotherapy, more frequently associated with a negative MRD (minimal residual disease, defined as less than 1 CLL cell in 10,000 leukocytes) and prolonged PFS (median PFS 27.6 and 16.3 months vs. 11.1 months, respectively) [20].Since obinutuzumab showed higher potency, equal tolerability and sparely enriched adverse effects than rituximab, it was FDA-approved in 2013 for untreated CLL patients in combination with chlorambucil who are not eligible for a high intensive therapy. Infection rates of obinutuzumab were similar to rituximab, but more severe infusion-related side effects were observed.Nevertheless, rituximab is the only CD20 antibody which is broadly approved for combined use, while obinutuzumab is currently approved in combination with clb or with venetoclax (see below). ",
"section_name": "Chemoimmunotherapy in CLL",
"section_num": null
},
{
"section_content": "B cell receptor (BCR) signaling is essential for CLL cells.Several novel agents target molecules of the BCR pathway and are effective even in high-risk CLL.In 2014, the FDA approved ibrutinib, an orally bioavailable BTK (Bruton's tyrosine kinase) inhibitor, as monotherapy in relapsed/refractory CLL (R/R).The mode of action of ibrutinib operates via specific covalent binding to the cysteine 481 in the active site of the BTK enzyme.Binding to BTK inhibits downstream signaling such as MAPK, PI3K, and NF-kB, and reduces migration and proliferation of the tumor cell [21].In patients, ibrutinib induces lymphocytosis with an asymptomatic increase of tumor cells in the peripheral blood, whereas rapid shrinka g e o f l y m p h n o d e s a n d s p l e e n i s o b s e r v e d .Lymphocytosis alone must not be correlated with disease progression and usually resolves within a few months of therapy [22].Approval of ibrutinib was based on a phase Ib/II trial, where 85 heavily pretreated R/R patients reached a PFS of 75% and OS of 83% after 26 months [23•].Intriguingly, the response was independent of genomic risk factors, prior therapies, or the presence of del(17p).A 5-year follow-up of an extended patient collective showed an overall response of 92% in treatmentnaïve patients and 89% in R/R patients, respectively.The PFS rate of ibrutinib monotherapy was 92% in treatmentnaïve patients and 44% in R/R patients, while the OS rate was 92% and 60%.Hematological adverse events (AEs) such as grade 3 cytopenia, neutropenia, and thrombocytopenia decreased over time [24]. The phase III RESONATE study compared single-agent ibrutinib and ofatumumab in high-risk relapsed patients.Follow-up after only 19 months showed a significantly longer PFS in the ibrutinib arm with 91% of patients attaining a response.The PFS at 24 month was 74%.Long-term follow-up reported a continuous response to ibrutinib, which was increasing over time.Median duration of ibrutinib was 41 months, with 46% remaining on treatment at a median follow-up of 44 months [25]. The phase III HELIOS trial compared ibrutinib and bendamustine with rituximab in treated CLL without del(17p).At an observation time of 18 months, BR + ibrutinib were superior with a PFS of 79% in comparison with 24% in the BR + placebo group.Impressively, the PFS at 36 months was 68% vs 13.9% (Fig. 1a).Overall survival at 36 months was 81.6% vs. 72.9%,(Fig. 1b) [26]. In the front line treatment setting, the RESONATE-2 phase III study compared ibrutinib vs. chlorambucil in elderly, untreated patients.A superior response to ibrutinib was demonstrated with a PFS of 24 months, whereas the median was not reached under treatment with chlorambucil as compared with 18.9 months under ibrutinib and an OS of 85% vs. 98.9%, respectively [27]. More recent clinical phase III trials demonstrated superiority of ibrutinib in comparison with chemoimmunotherapy (ECOG-ACRIN E1912, Alliance A041202, iILLUMINATE) [28][29][30]. The phase III E1912 trial demonstrated superior PFS and OS for ibrutinib + rituximab in comparison with FCR in a large cohort of 510 untreated young and fit patients without del(17p) [28].Alliance A041202 compared ibrutinib with ibrutinib + rituximab and bendamustine + rituximab (BR).Ibrutinib monotherapy or ibrutinib + rituximab prolonged PFS in comparison with BR as a frontline regimen.Of note, the addition of rituximab did not add a benefit in comparison with ibrutinib monotherapy [29].With iLLUMINATE, the chemotherapy free combination of ibrutinib + obinutuzumab was validated to be superior in naïve patients compared with chlorambucil + obinutuzumab [30]. Based on these extensive clinical trial data, ibrutinib is the preferred option as first line therapy in old and young patients.One disadvantage of the regimen is the need for continuous therapy, which may stimulate resistance generation due to continued selection pressure. Despite its initial efficacy, patients relapse under ibrutinib therapy.Interestingly, analysis of tumor cells of patients who progressed under ibrutinib therapy revealed specific mutations in BTK at position 481.Of importance, the C481S mutation of BTK conferred resistance by preventing the covalent binding of ibrutinib to its target cysteine 481 in BTK [31, 32•].Furthermore, several mutations were identified in PLCG2, which functions downstream of BTK.The gain-of function mutations in PLCG2 induce hyperreactive BCR signaling mediated by RAC2 and loss of dependence on BTK [33].Sequencing of relapsed patient cells uncovered that 85% of all patients which relapse during ibrutinib treatment, carried a BTK or PLCG2 mutation.The mutations were already detectable early after a median of 9.4 months of ibrutinib treatment.This knowledge is essential to avoid early resistance and could help to decide a possible switch of interventions [34]. ",
"section_name": "Targeting BCR Signaling",
"section_num": null
},
{
"section_content": "The phosphoinositide 3-kinase (PI3K) delta inhibitor idelalisib targets BCR signaling in CLL cells.It was FDA/ European Medicines Agency (EMA)-approved in 2014 for patients with refractory CLL.The approval was based on a phase III study in which idelalisib and rituximab significantly improved PFS and OS [35].Idelalisib also showed efficacy in patients with del(17p) or TP53 mutations.Furthermore, interim results of a phase III trial demonstrated that the combination of bendamustine, rituximab, and idelalisib significantly enhanced PFS in patients with R/R CLL in comparison with BR + placebo (median PFA 20.8 vs. 11.1 months) [36].Very recent updates of the Gilead 116/117 trial report improved efficacy of rituximab/idelalisib compared with rituximab monotherapy: an overall response rate (ORR) of 85.5% after 20.3 months of follow-up.Improved OS was the greatest among patients with del(17p) or TP53 mutation.Additionally, IGHV unmutated cases showed similar efficacy.In contrast, long-term exposure to idelalisib increased adverse effects, especially grade 3 diarrhea, colitis, and pneumonitis [37]. At the beginning of 2018, the approval of idelalisib in combination with rituximab and bendamustine for R/R CLL was withdrawn because of toxicity concerns.Black boxed warnings on idelalisib drug treatment were implemented due to fatal or serious hepatotoxicity (11-18%), %), diarrhea/ colitis (14-19%), pneumonitis (4%), infections (21-36%), intestinal perforation, and transaminitis (54% grade 3) [38].An interim analysis of three earlier-stage clinical trials demonstrated decreased overall survival in the idelalisib arms due to severe infections with PJP (pneumocystis jiroveci fungus) and CMV (cytomegalovirus).The EMA mandated additional safety measurements, PJP prophylaxis and CMV monitoring.Preclinical research data demonstrate that PI3K inhibition can modulate the effect in regulatory T cells on tumor cells, supporting the effect of PI3Kδ inhibitors.Nonetheless, CD8+ T cell function was decreased upon PI3K inhibition which might explain increased susceptibility for infections [39].Acquired resistance to idelalisib treatment has been observed in humans, although no unique recurrent mutation was identified [40].Preclinical data show that resistance to PI3Kδ inhibition does not rely on a unique mutation, though resistance to PI3Kδ inhibition induces a relevant activation of IGF1R, resulting in enhanced MAPK signaling [41]. Since idelalisib treatment appears to be associated with a higher rate of adverse effects than ibrutinib or venetoclax, idelalisib is not selected as the first choice for relapsed/ refractory patients and remains reserved for higher lines of therapy and for patients not suitable for other therapeutic options. Duvelisib (IPI-145) is the second FDA-approved PI3K inhibitor, targeting γ and δ isoforms.Preclinical studies demonstrate greater activity of inhibition of both isoforms γ and δ than PI3Kδ alone [42].The phase III DUO trial compared duvelisib monotherapy with ofatumumab in patients with R/R CLL.Patients treated with duvelisib had significantly better PFS and ORR.Serious adverse effects were reported in 67% of patients treated with duvelisib.Adverse effects grade 3 occurred in 87% of the duvelisib treated patients compared with 48% in the ofatumumab-treated arm.Pneumonia, infections, and diarrhea were the most frequent [43].Based on the DUO trial, duvelisib was FDA-approved for R/R CLL after two prior lines of therapy in 2018. ",
"section_name": "PI3K Inhibitors Idelalisib and Duvelisib",
"section_num": null
},
{
"section_content": "A novel treatment principle was introduced by targeting B cell lymphoma 2 (BCL2) using the BH3 mimetics, which cause immediate apoptosis of tumor cells.BCL2 family proteins play a major role in the regulation of cell death and are highly conserved.The BCL2 family is clustered into three main functional groups, the pro-survival and anti-apoptotic proteins BCL2, MCL-1, BCL-x L , and BCL-w [44][45][46], the multi-BH domain pro-apoptotic proteins BAX and BAK, and the pro-apoptotic BH3-only proteins BIM, tBID; BAD, PUMA; NOXA, and HRK [47][48][49] that trigger and execute the \"suicidal\" cell death.In healthy cells, the balance between cell survival and cell death requires dynamic binding interactions between pro-apoptotic and anti-apoptotic proteins. C L L ce l l s o v e r e xp r e s s a nt i -a p o p t o t i c B C L 2 .Overexpression of BCL2 is caused by various mechanisms.The most common cytogenetic abnormality is the del(13q14), the minimally deleted region of which includes the BCL2 repressors and microRNAs 15 and 16 [50].Moreover, hypomethylation of BCL2 in CLL also contributes to BCL2 upregulation due to epigenetic dysregulation [51,52].On the other hand, defects in expression of pro-apoptotic members result in a loss of the tumor suppressive function and lead to an imbalance between pro-and anti-apoptotic BCL2 family proteins.Homozygous deletions or inactivating mutations of BAX and BID [53,54] or defective expression of BID and PUMA due to loss of p53 function also tip the balance towards antiapoptotic proteins [55,56]. BCL2 can be selectively targeted with venetoclax which is a novel, orally bioavailable BH3 mimetic.Venetoclax shows high efficacy in particular in the treatment of CLL, but preliminarily also in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL).BH3 mimetics bind to the hydrophobic groove of BCL2 inducing apoptosis.The precursor molecule of venetoclax, ABT-737 had a binding potential to BCL2, BCL-x L , and BCL-w, and showed activity in vitro.Navitoclax (ABT-263), an orally available derivate, showed promising efficacy in CLL patients [57]; however, strong inhibition of BCL-x L induced a rapid decrease in circulating platelets and thrombocytopenia which arrested its clinical development. Venetoclax was approved by the FDA and EMA in December 2016 for patients with previously treated CLL with del(17p13) and patients failing B cell receptor signaling inhibitors (EMA only), or who carry a TP53 mutation and are refractory to chemoimmunotherapy and BCR inhibitors recruited in the phase II M13-982 and M14-032 trials [58•].M13-982 was a pivotal phase II clinical trial, enrolling relapsed/refractory CLL patients with del(17p).In this multicenter open-label study, 158 patients were treated with venetoclax with a weekly dose ramp up from 20 to 400 mg over 4 weeks (20,50,100,200, and 400 mg, due to the risk of tumor lysis syndrome (TLS), see below) which was continued until disease progression.At a median time of 26.6 months on study, 77% of all patients had achieved an ORR (122 of 158 patients).The 24-month estimate for ongoing response was 66% (95% CI, 55% to 74%, Fig. 2a); the 24-month estimate of PFS and OS were 54% (95% CI, 45% to 62%, Fig. 2b), and 73% (95% CI, 65% to 79%, Fig. 2c), respectively.Estimated PFS of patients with complete remission (CR) or incomplete bone marrow recovery (CR i ) or nodular partial remission (nPR) at 27.2 months was not reached (Fig. 2d) [59]. Responses of venetoclax treatment were durable, and a majority of the patients showed a reduction in absolute lymphocyte count, lymph node lesion diameter, and bone marrow infiltrate at a median of 0.3 months of treatment.Management of the tumor lysis syndrome occurred using prophylaxis in 6 patients; none of them reached clinical TLS.Hematologic adverse effects of higher grade (grade ≥ 3) were neutropenia (42%), anemia (25%), and thrombocytopenia (20%).The results of this pivotal trial led to FDA approval of venetoclax in April 2016 for the treatment of previously treated CLL patients with del(17p) [60•]. M14-032 recruited relapsed/refractory patients after previous treatment with ibrutinib or idelalisib.The ORR in the ibrutinib pretreated group was 65%, the 12-month PFS was 75%.In the idelalisib-pretreated group, the ORR was 67%, and an estimated 1-year progression-free survival of 79% was found [61].Most common grade 3 or 4 adverse effects were neutropenia (51%), thrombocytopenia (29%), and anemia (29%).Of 91, 17 patients died because of progressive disease [61].Importantly, venetoclax induced deep remissions with MRD negativity in contrast to treatment with BCR pathway inhibitors [62].No incidence of tumor lysis syndrome (TLS) was observed. Since the application of venetoclax induces a rapid reduction of tumor cells with abrupt onset within 6-8 h after dosing, TLS is a major risk issue in clinical care, owing to the high potency of the drug.Dimension of TLS, resulting in rapid cell death, is dependent on tumor mass [63,64], comorbidities such as renal function, and treatment dose [63].Quick release of metabolites into the blood stream destabilizes renal excretion, which causes hyperuricemia, hyperkalemia, hyperphosphatemia, and hypocalcemia, following acute renal failure and cardiac events of life-threatening potential.For this reason, patients in early treatment phase should be monitored closely for TLS.Prophylaxis of TLS including hydration, diuresis, monitoring of electrolytes, and prevention of hyperuricemia should be performed according to its severity and treatment following the guidelines on risk assessment and prophylaxis of TLS [65]. Despite high rates of durable remission in patients, continuous daily treatment with venetoclax induces secondary resistance.Venetoclax resistance was shown to be implicated with complex clonal shifts [66].Using an in vitro genome-scale screen, Guièze et al. identified regulators of lymphoid transcription and cellular metabolism as resistance drivers.Confirmation in CLL patient cells with early progressive disease uncovered MCL-1 overexpression and AMPK signaling responsible for venetoclax resistance [66].Whole exome sequencing of 8 CLL patients with dysfunctional TP53 and a median time of 15.4 months from initial treatment until progression uncovered heterogeneous clonal evolution under venetoclax treatment.Two patients developed a BTG1 mutation; 3 patients had a homozygous deletion affecting CDKN2A/B, one BRAF mutation, and an amplification of CD274 were deciphered [67].Very recently, another recurrent mutation was reported by comparison of paired prevenetoclax and progression samples after long-term treatment.Seven of 15 patients carried a novel Gly101Val mutation in BCL2, which was selected at progression after 19 to 42 months, but not at the beginning of treatment [68].The authors showed that Gly101Val reduces the affinity of BCL2 for venetoclax by 180-fold.In a second report, Tausch et al. identified the BCL2 Gly101Val mutation and an additional D103Y mutation in 3 of 4 venetoclax progressive patients [69].These data demonstrate a selection of cell clones which acquire mutations under selection pressure of venetoclax.Especially, long-term treatment is prone to drive the evolution of resistant tumor cells.Clinical trials therefore aimed at timelimited, highly effective combination strategies to obtain deep remissions and to prevent secondary resistance generation. The MURANO trial was the pivotal trial establishing timelimited therapy with venetoclax and led to the extension of the FDA and EMA approval of venetoclax and rituximab for all R/R CLL patients, regardless their del(17p) status.This phase III trial aimed to test efficacy and safety of the combination venetoclax and rituximab (VR) in order to avoid secondary resistance generation.In brief, 194 rel/ref.patients were treated 2 years with venetoclax and received 6 cycles of rituximab, compared with 6 cycles of bendamustine-rituximab (BR).At 24 months, PFS was estimated to be 84.9%compared with 36.3%, respectively.MRD negativity, OS, and ORR were impressively improved.After termination of venetoclax, follow-up at 3 years demonstrated an excellent benefit of VR against BR in regard to PFS (71.4% vs. 15.2%) with slightly enriched adverse effects (neutropenia 57.7% vs. 38.8%) in the VR group [70]. In May 2019, the FDA approved venetoclax for front line CLL treatment based on the CLL14 study [71].Four hundred thirty-two patients with previously untreated CLL and coexisting medical conditions were randomized to venetoclax and obinutuzumab (Ven + G) or obinutuzumab + chlorambucil (GClb).After a follow-up of 24 months, the Kaplan-Meier estimate of the percentage of PFS was significantly higher in the Ven + G group than in the GClb group (88.2% vs. 64.1%);PFS was still significantly improved at 28.1 months in the Ven + G group (30 primary end-points counted vs. 77).Moreover, patients with TP53 deletion, mutations, or unmutated IGHV had a significant benefit as well.Adverse effects as grade 3 or 4 neutropenia or infections were comparable in both of the arms [71]. ",
"section_name": "Targeting B Cell Lymphoma 2 (BCL2)",
"section_num": null
},
{
"section_content": "Venetoclax and ibrutinib have two different modes of action, and preclinical studies in human and mouse CLL cells demonstrated synergistic activity [72,73].Efficacy profiles of the two drugs behave also complementarily, since ibrutinibdependent lymphocytosis leads to clearance of tumor cells from lymph nodes and mobilizes them into the peripheral 2018;36:1973-1980 [59] blood.In contrast to ibrutinib, venetoclax-induced apoptosis has prominent efficacy in the blood and marrow.Moreover, venetoclax is able to induce MRD negativity, which rarely occurred with ibrutinib treatment.Thus, the CAPTIVATE trial analyzed the combination of ibrutinib and venetoclax in 163 treatment naïve CLL.In the combination arm, patients received single-agent ibrutinib for the first 3 cycles (cycle = 28 days) followed by an ibrutinib plus venetoclax combination for at least 12 cycles.After MRD negativity, ibrutinib was continued daily.Early analysis shows a CR rate of 100% as well as MRD negativity of 82% [74]. Following a similar strategy, the ongoing phase II CLARITY trial aims to determine therapeutic activity and safety of this combination with the intention to stop therapy.An interim result demonstrated that the primary endpoint, MRD negativity, was achieved in 28/53 (53%) patients in PB and 19/28 (36%) in bone marrow.Impressively, 89% patients responded, and CR rate was 51%, ongoing with high tolerance and acceptable adverse events [75]. Another phase II study of combined ibrutinib and venetoclax involved 80 naïve high-risk and older patients.Patients carried at least a high-risk feature (del(TP53) or TP53mut, del(11q), or unmutated IGHV).Impressive responses across all subgroups in the subset of patients who completed 12 cycles of treatment with 88% of complete remission and 61% remission with undetectable MRD [76]. Further trials which are still ongoing are the NCT02756897 (combined treatment of ibrutinib and venetoclax), NCT03226301 and NCT03045328 (combined treatment of ibrutinib and venetoclax in R/R patients), NCT03128879 (high risk CLL), and NCT03513562 (patients with ibrutinibresistant mutations). Further development aiming at deep responses are triple therapies with ibrutinib, venetoclax, and obinutuzumab (NCT02427451, NCT02758665/CLL2-GIVe, and NCT02950051/CLL13). NCT02427451 was reported in 2018 by Rogers et al.In the phase 1b study, a small cohort of 12 R/R patients received 4 doses of obinutuzumab up to 8 courses.In parallel, they were treated with ibrutinib daily starting on course 2 and venetoclax, starting at course 3 for a total of 14 courses.To minimize risk of TLS, ibrutinib and venetoclax were introduced sequentially.The overall response rate was 92% with 6 patients being MRD negative at the end of treatment.Notably, no clinical or laboratory TLS occurred.Of the patients, 33% sustained grade ≥ 3 neutropenia, which is comparable with single treatment of venetoclax [77]. The phase 2 CLL2 BAG trial aimed to first reduce tumor cells using bendamustine followed by maintenance therapy by obinutuzumab and venetoclax up to 24 months.A total of 66 patients were enrolled and treated with the sequential triple T concept.First, the 63 patients (34 treatment naïve and 29 R/R) received bendamustine for the debulking of tumor cells.After two 28-day cycles, maintenance therapy with obinutuzumab and venetoclax in a weekly dose escalation was proceeded.To date, most common adverse effects were neutropenia (44%), infections (14%), and thrombocytopenia (9%) [78]. The primary objective of the multicenter phase II CLL2-GIVe trial aims to evaluate the efficacy of ibrutinib and venetoclax and obinutuzumab in physically fit or unfit, previously untreated patients with del(17p) or TP53 mutation [79] Currently, all 41 high-risk patients have been recruited, and the primary endpoint (CR rate after 12 cycles of treatment) will be available in Q2 2020. The phase III CLL13 trial (GAIA), an international four arm study for physically fit patients, is testing chemotherapy-free frontline therapy for previously untreated patients without del(17p).The trial is fully enrolled and tested with standard chemotherapy (FCR/BR), venetoclax plus rituximab (RVe), venetoclax plus obinutuzumab (GVe) and venetoclax plus ibrutinib, and obinutuzumab (GIVe) with MRD and PFS as co-primary endpoints [80]. ",
"section_name": "Combination Trials of BCR Inhibitors and BCL2 Inhibitors",
"section_num": null
},
{
"section_content": "Ibrutinib and venetoclax monotherapy have been initially licensed based on remarkable single-agent efficacy and favorable tolerability profile, including chemo-refractory and genetic high-risk subgroups of CLL.However, resistance due to specific mechanisms (e.g., BTK, PLCg2, and BCL2 mutations) is emerging indicating the need to develop time-limited combinations.Approved combination treatments such as venetoclax or ibrutinib with obinutuzumab show high response rates and long remission durations.It is possible that these strategies may lead to long-term remission and potential cure of CLL.Comparison of sequential treatment with combinations and discontinuation of therapy are important aspects which need to be investigated. Based on the high rate of deep responses and long remission durations combined with good tolerability, venetoclaxbased combinations with BTK inhibitors or triple combinations are very likely the future of CLL treatment; however, the approaches are still experimental and require more clinical data before adapted in general practice. Funding Information Open Access funding provided by Projekt DEAL. ",
"section_name": "Conclusion",
"section_num": null
},
{
"section_content": "Conflict of Interest Annika Scheffold declares that she has no conflict of interest. Stephan Stilgenbauer has received fees for consulting, drug/equipment supplied by the entity, travel paid by the entity, and provided writing assistance for AbbVie, AstraZeneca, Celgene, Gilead, GlaxoSmithKline, Hoffmann-La Roche, Janssen, Novartis, Pharmacyclics, and Sunesis Pharmaceuticals. Human and Animal Rights and Informed Consent This article contains studies with human subjects which has been published by the authors. 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://creativecommons.org/licenses/by/4.0/. ",
"section_name": "Compliance with Ethical Standards",
"section_num": null
}
] |
[] |
10.1371/journal.pone.0164660
|
PlexinD1 Is a Novel Transcriptional Target and Effector of Notch Signaling in Cancer Cells
|
The secreted semaphorin Sema3E controls cell migration and invasiveness in cancer cells. Sema3E-receptor, PlexinD1, is frequently upregulated in melanoma, breast, colon, ovarian and prostate cancers; however, the mechanisms underlying PlexinD1 upregulation and the downstream events elicited in tumor cells are still unclear. Here we show that the canonical RBPjk-dependent Notch signaling cascade controls PlexinD1 expression in primary endothelial and cancer cells. Transcriptional activation was studied by quantitative PCR and promoter activity reporter assays. We found that Notch ligands and constitutively activated intracellular forms of Notch receptors upregulated PlexinD1 expression; conversely RNAi-based knock-down, or pharmacological inhibition of Notch signaling by gamma-secretase inhibitors, downregulated PlexinD1 levels. Notably, both Notch1 and Notch3 expression positively correlates with PlexinD1 levels in prostate cancer, as well as in other tumor types. In prostate cancer cells, Sema3E-PlexinD1 axis was previously reported to regulate migration; however, implicated mechanisms were not elucidated. Here we show that in these cells PlexinD1 activity induces the expression of the transcription factor Slug, downregulates E-cadherin levels and enhances cell migration. Moreover, our mechanistic data identify PlexinD1 as a pivotal mediator of this signaling axis downstream of Notch in prostate cancer cells. In fact, on one hand, PlexinD1 is required to mediate cell migration and E-cadherin regulation elicited by Notch. On the other hand, PlexinD1 upregulation is sufficient to induce prostate cancer cell migration and metastatic potential in mice, leading to functional rescue in the absence of Notch. In sum, our work identifies PlexinD1 as a novel transcriptional target induced by Notch signaling, and reveals its role promoting prostate cancer cell migration and downregulating E-cadherin levels in Slug-dependent manner. Collectively, these findings suggest that Notch-PlexinD1 signaling axis may be targeted to impair prostate cancer cell invasiveness and metastasis.
|
[
{
"section_content": "Plexins are cell surface receptors for extracellular signals of the semaphorin family [1].Mammalian semaphorin genes are divided into five classes-Class 3 to 7, which bind directly to Plexins or in association with co-receptor molecules.Semaphorin signaling has been implicated in a wide range of functions in development and in disease, ranging from axon guidance during morphogenesis to cancer progression [2].Semaphorin signaling has been found to regulate multiple hallmarks of cancer such as invasion, angiogenesis, and proliferation among others [3].Depending on the context of available signaling intermediates, they are found to play either a tumor suppressive or a pro-tumorigenic role.For instance, in endothelial cells Sema3E and its specific receptor PlexinD1 inhibit cell-substrate adhesion [4] and exert an anti-angiogenic function, while in cancer cells they have been shown to have a pro-tumorigenic role [5].In particular, Sema3E and its mature isoform Sema3E-p61 regulate migration and invasion of melanoma, colon, lung, and ovarian cancer cells, and Sema3E-PlexinD1 signaling was reported to promote invasive/metastatic phenotype [5,6].We reported previously that in certain cancer cells Sema3E-PlexinD1 can transactivate ErbB2 signaling, promoting the invasive/metastatic phenotype [5].Other studies showed Sema3E-dependent activation and nuclear translocation of the transcription factor Snail in ovarian cancer cells [6], or rather dependence-receptor features of PlexinD1 in breast cancer cells [7]. Notably, PlexinD1 is expressed at low levels in adult tissues, but it is typically overexpressed in multiple types of human cancer [5,6,7,8,9], both in tumor cells and in tumor vasculature; yet, the mechanisms sustaining this expression have not been elucidated.PlexinD1 is also remarkably expressed in endothelial cells, where it is required for vascular patterning in angiogenesis [10].Recently, it was reported that VEGF positively controls the expression of PlexinD1 in endothelial cells of actively sprouting blood vessels in retinal development [11].Moreover, Plex-inD1 expression was induced by hypoxia in post-ischemic regenerating vessels [12].However, the relevance of this pathway in other tissues has not been investigated.Notably, in the vasculature of developing retina Notch signaling was associated in one study with reduced PlexinD1 expression [11], while in a different developmental system this was not confirmed [13].There are four known Notch receptors (Notch1-Notch4); upon binding of ligands of the DSL family -Jag1, Jag2, Dll1, Dll3, Dll4-Notch receptors undergo subsequent cleavage by TACE and gamma secretase.Once released in the cytosol, Notch intracellular domain (N-ICD) translocates into the nucleus, where it forms complex with RBPjk/CBF1 transcription factor to regulate gene transcription.Notch family members are overexpressed in several human tumors, and their signaling cascade is often activated by an increased load of notch ligands in the tumor microenvironment, for example in response to inflammation or hypoxia [14,15].Sema3E-PlexinD1 signaling was recently reported to be upregulated in prostate cancer and regulate cell migration [8].Prostate cancer is one of the most frequently diagnosed cancers in men.While relatively tractable in its early stage, in the advanced stage this cancer has a poor prognosis due to regional invasion and metastatic dissemination.The implicated mechanisms are under intense investigation.Similar to other tumors, in prostate cancer, Sema3E was found to be expressed at higher levels compared to normal tissue; PlexinD1 staining too was found to be higher in tumors compared to benign tissue [8].It was further observed that the cleaved form of Sema3E, Sema3E-p61, was expressed in prostate cancer cell lines.However, the mechanisms regulating cell migration downstream of PlexinD1 in this system are still unclear.On a separate note, Notch signaling ligand-Jag1-has been independently reported to be a marker of poor prostate cancer progression [16].Consistently, Jagged1-Notch1 signaling has been shown to regulate migration and invasion of prostate cancer cells via the activation of Akt, mTOR and NF-kB signaling pathways [17]. Here we report that the expression of Sema3E-receptor PlexinD1 is under control of the Notch pathway, which is commonly activated in tumors and associated with malignant progression.PlexinD1 regulation occurs at the promoter level, through the canonical Notch signaling pathway mediated by RBPjk/CBF1 transcription factor.Notch signaling cascade has been found to promote cancer metastasis via transcriptional programs regulating cell-cell contact and cell migration [18,19].Here we show that the upregulation of Notch-target PlexinD1 in prostate cancer cells upholds Slug expression and downregulates E-cadherin levels, a program associated with cell migration, epithelial-to-mesenchymal transition and metastasis. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "Tumor cell lines were obtained from ATCC and cultured in a humidified incubator with 5% CO 2 at 37°C in media supplemented with 10% FBS, penicillin and streptomycin.U87MG, U251, A549 were cultured in DMEM; PC3 and DU145 were cultured in RPMI; SKOV3 was cultured in McCoy media; HUVEC were cultured in EGM-2 media containing growth factors (Clonetics), 100 IU/ml of penicillin G sodium and 100ug/ml of streptomycin sulfate.Cell treatments with gamma secretase inhibitors DAPT (Sigma, D5942-5MG; used 25μM) and RO4929097 (Selleckchem, S1575; used 25μM) were done for 72 hours in media supplemented with 10% FBS.Cell treatments with LY294002 (10μM) and PD98059 (10μM) were done for 24 hours in media supplemented with 10% FBS. ",
"section_name": "Cell lines and chemicals",
"section_num": null
},
{
"section_content": "Stable cDNA and shRNA expression in mammalian cells was achieved by means of lentiviral vectors, as previously described [20].Briefly, calcium phosphate method was used to co-transfect transfer plasmids, packaging vectors and constructs expressing VSV-G envelope protein in 293T cells.Tumor cells were transduced by incubation with lentiviral vector suspensions, in the presence of 8μg/ml polybrene, for 8-12 hours.In other experiments, cDNA and siRNAexpressing constructs were transiently transfected with Lipofectamine 2000 (Life Technologies) according to manufacturer's instructions. ",
"section_name": "Gene transfer and RNA interference in mammalian cells",
"section_num": null
},
{
"section_content": "Notch1 downregulation was achieved by lentiviral-mediated stable expression of (puromycin selectable) shRNA expression constructs: TRCN0000350330, TRCN0000003359 or TRCN0000003362 (indicated as shNotch1_2, shNotch1_3, and shNotch1_5, respectively in the validation experiment shown in S3C Fig); the latter construct was also simply indicated as shNotch1 in experiments throughout the manuscript.PlexinD1 downregulation was achieved by (puromycin selectable) lentiviral-mediated stable expression of shRNA TRCN0000061548 (#48; mainly used in this study and usually dubbed as \"shPlexinD1\" in the manuscript) and TRCN0000061552 (#52); or by transfection of siRNAs targeting the 3'-UTR sequence of PLXND1 transcript: GCUACUUGAUCUUGCUGAA. Sema3E knock down was achieved by stable expression of shSema3E sequence: GGTTACGCCTGTCA CATAA [5].Lentiviral vectors were also used to transfer stable expression of VSV-G tagged PlexinD1 (simply dubbed as \"PlexinD1\" in the manuscript), as previously shown [5].Constitutively active intracellular domains of Notch1 and Notch3 (N-ICD), or truncated Notch1-ΔE construct [21] (a gift from Claudio Talora and Isabella Screpanti, Rome) were overexpressed by transient transfection.Mouse Jag1 Fc and Dll1 Fc cloned in pTracer CMV vectors [22] were gifts from Takayasu Kato.Hes1-Luc reporter construct, cloned in pGL2 [23] was a gift from Alain Israel, France.12XCBF/12XCSL-DsRedExpressDR reporter construct was a gift from Federico Bussolino and Urban Lendhal [24].Slug (SNAI2 gene) silencing was achieved by transfection of siRNA sequence: 5 0 -CAAUAAGACCUAUUCAACU -3 [25]. ",
"section_name": "siRNA, shRNA and DNA constructs",
"section_num": null
},
{
"section_content": "Luciferase reporter assays were done with Hes1 promoter driving firefly luciferase or with a 1567bp region of PlexinD1 promoter (from 1388bp upstream of TSS to 179bp downstream of it) (Genecopoeia) driving secretable Gaussia luciferase.Cells were transfected using lipofectamine 2000 with 1ug of reporter construct in combination with 10-100ng of Notch1-intracellular domain (N1-ICD), N3-ICD, RBPjk1, or DN-RBPjk expressing constructs; a GFPexpressing plasmid was added in all conditions, for internal normalization purposes.48hrs after transfection, cells were lysed and luciferase assays conducted using luciferase assay system (Promega, Madison, WI, USA).For Hes1-Luc assay, Promega luciferase assay system was used; for PlexinD1-Luc assay, Secrete-Pair Gaussia Luciferase assay system (Genecopoeia) was used.Two mutants of PlexinD1 Gaussia Luc reporter were generated using restriction site-based deletion: Mut1_D1 was deleted between EcoR1 and AfeI sites in the promoter sequence, while Mut2_D1 derived from deletion of the sequence comprised between two NheI sites (both constructs were verified by sequencing). ",
"section_name": "Luciferase reporter assays",
"section_num": null
},
{
"section_content": "Transwell migration assays were performed using Transwell 1 chamber inserts (Costar, Cambridge, MA) with a porous polycarbonate membrane (8 μM pore size; Corning Costar Incorporated, NY, USA).Briefly, the lower side of the filter was coated with 10 μg/ml fibronectin and blocked with 1% BSA.Approximately 5x10 4 cells were added in the upper chamber, and allowed to migrate through the filter towards the lower chamber containing the indicated factors.In parallel, the equal number of cells were seeded in cell culture multiwell plates to check for equal cell loading.After 24hrs non-migrated cells from the upper side were removed by a cotton swab, followed by fixing of cells with 11% Glutaraldehyde and staining with crystal violet.Microscopic images were then quantified either by cell counting or by converting to a binary image and quantifying the integrated pixel values using ImageJ (NIH).Experiments were repeated at least twice in replicates, showing consistent results. ",
"section_name": "Transwell migration assays",
"section_num": null
},
{
"section_content": "Fluorescent labeling of cells was done using Vybrant DiD cell labeling solution (Molecular Probes).Cells were washed with PBS and incubated with 10μM Vybrant DiD in 10% FBS RPMI media for 30 mins, allowing the cells to get labelled.Cells were then trypsinized, centrifuged and washed twice with PBS.Cells were then counted and suspended in PBS and used in extravasation assay. 1 million cells were used per mice. ",
"section_name": "Fluorescence labeling of living cells",
"section_num": null
},
{
"section_content": "Wound healing assay was performed in confluent monolayer of cells grown in 6-well plates.A pipette tip was used to make three scratches in cell monolayers; cells were washed twice and images were taken at starting time point, followed by incubation in appropriate media.Later images were taken after 24 hrs, images were aligned and analyzed to score for wound closure (based on measurement of residual wound area).In the case of DAPT and RO4929097 treatments, cells were pre-incubated with the drugs for 48hrs. ",
"section_name": "Wound healing assay",
"section_num": null
},
{
"section_content": "Total RNA from tumor cell lines was isolated using RNeasy Protect Mini kit (Qiagen) according to the manufacturer's instructions.cDNA preparation was performed according to standard procedures, using M-MLV Reverse Transcriptase (Promega) and oligo-dT primers / random hexamers.PCR was performed by applying Taqman probes (Applied biosystems): PlexinD1 (Hs00391129_m1, Applied biosystems), Notch1 (Hs01062014_m1), Hes1 (Hs00172878_m1), beta actin (Hs99999903_m1).Alternatively, PCR was conducted with SYBR Green Master Mix (Life Technologies) and run in Applied Biosystems 7900HT Fast Real-Time PCR system, by applying the following primer pairs: PlexinD1 (Fwd-ACCGAGCAGTGGATGATTCT, Rev-TCCTGGTGAACGACACAGAC), GAPDH (Fwd-GAAGGTGAAGGTCGGAGTC, Rev-GAA GATGGTGATGGGATTTC), PlexinB1 (Fwd-CACTGAACCCCACACCTTTC, Rev-ATAGCCACCACCTCCTCCTT), Snail (Fwd-GACTACCGCTGCTCCATTCCA, Rev-TCCTCTTCATCACTAATGGGGCTTT), Zeb2 (Fwd-TTCCATTGCTGTGGGCCT, Rev-TTGTGGGAGGGTTACTGTTGG), Slug (Fwd-AGATGCATATTCGGACCCAC, Rev-CCTCATGTTTGTGCAGGAGA), Zeb1 (Fwd-AGCAGTGAAAGAGAAGGGAATGC, Rev-GGTCCTCTTCAGGTGCCTCAG), Twist1 (Fwd-TGTCCGCGTCCCACTAGC, Rev-TGTCCATTTTCTCCTTCTCTGG). ",
"section_name": "Real time quantitative PCR analysis of gene expression",
"section_num": null
},
{
"section_content": "Metastatic tumor cells were labelled in culture by incubation with Vybrant DiD (Life Technologies), according to manufacturer's specification.One million fluorescent-labelled PC3 cells were then injected into the lateral tail vein of 6-8 weeks old NOD/SCID mice (Charles River Laboratory); experimental groups included 5 animals each.The mice were sacrificed 48 hours after injection, and quantification of metastatic cells in the lungs was done by fluorescence microscopy by analyzing at least four independent microscopic fields per lung, using ImageJ software (NIH) to measure signal intensity.Mice were housed in individual ventilated cages.Animal handling was performed according to international guidelines for animal experimentation and the specific Italian Legislative Decrees no.116 (27 Jan 1992) ",
"section_name": "Metastatic cell extravasation assay",
"section_num": null
},
{
"section_content": "Total cell lysates were prepared in a Tris pH 6.8-10% SDS solution (1:1) by heating at 95°for 20 mins.Protein concentration was measured using Pierce BCA protein assay kit as per the company instructions.For western blotting 10-40 ug of protein was resolved on 7.5% or 10% mini gels from Biorad, transferred to nitrocellulose membrane using semi-dry method and immunoblotted.10% BSA was used for filter blocking in all conditions. The following primary antibodies were used: against Notch1, Slug and Actin (Santa Cruz Biotechnology), against PlexinD1 (R&D Systems), against Vinculin (Sigma), against Notch3 (Cell Signaling technology), against E-cadherin (BD Transduction laboratories). ",
"section_name": "Protein analysis",
"section_num": null
},
{
"section_content": "The portal assembled by the www.cbioportal.org[26] was used to analyze the correlation of expression of PlexinD1 with Notch signaling genes.We elaborated correlation graphs of prostate adenocarcinoma, colon and rectum adenocarcinoma, thyroid carcinoma, kidney renal clear cell carcinoma TCGA datasets (provisional); mRNA expression z-score threshold was set at +/-2 fold, and the category of ' All tumors' was chosen with input of PLXND1, NOTCH1, NOTCH3 and SNAI2.Plots of correlation were generated under the heading of 'Plots' -> 'two genes'.The heading of 'Mutual exclusivity' gave the p values, while the input of individual genes yielded the spearman correlation coefficient under the heading 'co-expression'.GEO-GSE54460 prostate cancer cell dataset was also analyzed for correlation between SNAI2 and PLXND1 genes, while multiple genes expression fold change (vs.control) were analyzed in GEO-GSE40403 endothelial cell dataset, upon Jag1 overexpression or Notch1 knock down. ",
"section_name": "Dataset analysis",
"section_num": null
},
{
"section_content": "All in vitro and in vivo experiments were performed at least two-three times.Representative results of qPCR experiments are shown.T-test was used to assess statistical significance, indicated in graphs as follows: ÃÃÃ (p<0.001),ÃÃ (p<0.01),Ã (p 0.05). ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "PlexinD1 overexpression in high-grade vs. normal tissues or low-grade human tumors has been reported in a number of studies [5,6,8].Previous data suggested that PlexinD1 may be induced in the vasculature in response to hypoxia or Vascular Endothelial Growth Factor (VEGF) [11,12], conditions which are also known to regulate cancer cells.In preliminary experiments we found that VEGF significantly induced PLXND1 mRNA expression in endothelial HUVEC cells, but not in a range of human carcinoma cells; moreover, we also failed to observe any significant upregulation of PlexinD1 levels in cancer cell lines exposed to low oxygen tensions (data not shown).These data suggested that PlexinD1 expression in tumor cells may be regulated by mechanisms different from those observed in endothelial cells. Interestingly, by data mining in gene expression datasets, we found that-among other genes-PLXND1 levels were induced almost 4-fold upon constitutive Notch1 activation in glioblastoma stem cells [27].Initially, we validated these raw data in two glioblastoma cell lines-U87MG and U251.Indeed, upon overexpressing a constitutively activated form of Notch1 (N1-ICD) we observed an appreciable increase of PlexinD1 protein levels (S1A and S1B Fig) .These findings in glioblastoma cell lines encouraged us to further study this novel regulatory mechanism in other cell types.Constitutive Notch signaling established by transfection of N1-ICD or a ligand-independent activatable receptor (Notch1-ΔE ICD [28]), significantly induced endogenous PlexinD1 mRNA and protein levels in 293T cells (Fig 1A and1C).In other experiments, we analyzed HUVEC endothelial cells, which abundantly express PlexinD1 in basal conditions, and subjected them to stable Notch1 knock down by shRNAs or blunted its signaling cascade with the gamma secretase inhibitor DAPT [29].In either case, interfering with the Notch pathway led to concomitant PlexinD1 downregulation at mRNA and protein level (Fig 1D Prompted by these findings, we analyzed the TCGA dataset of different human cancers to search any potential correlation between PlexinD1 expression and that of Notch family members.To this end, we determined the correlation in the expression of PlexinD1 with Notch1 and Notch3 in datasets of prostate, colon, thyroid and kidney cancers.As shown in S2A Fig, PlexinD1 showed a positive Spearman correlation coefficient with Notch1 levels, ranging from 0.43 (prostate cancer) to 0.75 (kidney renal clear cell carcinoma).Notably, Notch signaling is implicated in the invasiveness of prostate carcinoma cells, and its expression correlates with tumor grade [30]; moreover, Blanc et al. independently reported that PlexinD1 is upregulated in prostate cancer [8]. Another Notch family receptor, Notch3, displayed significant positive Spearman correlation with PlexinD1 in different tumor types (S2B Fig) .As expected, the constitutively active intracellular domains of both genes were similarly capable of activating the transcription of known target genes (S2C and S2D ",
"section_name": "PlexinD1 is a novel transcriptional target of Notch signaling",
"section_num": null
},
{
"section_content": "In order to know if Notch-dependent regulation of PlexinD1 widely occurs in cancer cells, we stably knocked-down Notch1 expression by shRNA in a range of cell lines derived from colon (KM-20, COLO741), prostate (PC3), lung cancer (A549) and melanoma (MDA435), and consistently observed PlexinD1 downregulation (S3A Fig) .Of note, we observed that the regulation of PlexinD1 was specific, as Notch-targeting shRNAs did not affect the expression of another plexin family member implicated in cancer progression-PlexinB1 (S3B Fig) .Notch1 signaling appeared to be particularly relevant in regulating PlexinD1 mRNA levels in prostate cancer cells PC3 and DU145, and this was confirmed at the protein level (Fig 3A and3B).Importantly, we identified three independent shRNA sequences that could efficiently silence PlexinD1 expression in prostate cancer cells (S3C Fig) , and found that all of them caused concomitant downregulation of PlexinD1 levels, clearly suggesting a mechanistic link.Notch signaling is basally present in normal and tumor cells, mainly due to autocrine/juxtacrine ligand activity.Inhibitors of gamma-secretases are widely used reversible inhibitors that achieve a compound inhibition of all Notch family members; these drugs include DAPT [29] and RO4929097 [33], which is also under assessment in clinical trials.We confirmed that these Notch signaling is activated in response to different members of the Notch-ligand family.For instance, the membrane-bound ligand Jag1 is overexpressed in human tumors and has been linked to metastasis [16].Thus we tested the activity of a Jag-derived soluble peptide capable of activating Notch signaling in cellular models [34].Interestingly, Jag1 peptide could similarly induce the expression of the canonical Notch target Hes1, as well as that of PlexinD1 in PC3 cells (S5A Fig) .Also, PlexinD1 promoter reporter activity was upregulated by Jag1 peptide, while it was completely blunted by the combined treatment with gamma secretase inhibitor RO4929097 (S5B Fig) .In another set of experiments, we transiently overexpressed either ",
"section_name": "Notch signaling is responsible for sustaining PlexinD1 expression in cancer cells",
"section_num": null
},
{
"section_content": "A number of studies have reported that upregulated Notch signaling is associated with invasion and metastasis in prostate cancer [37,38].Notably, PlexinD1 levels are similarly regulated in human prostate tumors and this signaling cascade controls prostate cancer cell behavior [8]; however, the underlying molecular mechanisms were not elucidated.In order to assess the functional relevance of Notch-PlexinD1 signaling axis in prostate cancer cells, we performed wound healing and Boyden chamber transwell migration assays with PC3 and DU145 prostate cancer cells.We tested the effect of gamma secretase inhibitors blocking pan-Notch signaling on PC3 migration, and consistent with previous data on DAPT treatment [29] we observed a significant reduction of migratory ability (Fig 4A).Then, we observed that both Notch1 and PlexinD1 silencing by shRNAs led to decreased migration in wound healing assays (Fig 4B PlexinD1 expression in prostate cancer cells.Yet, the molecular mechanisms by which Sema3E-PlexinD1 signaling could induce the migration of prostate cancer cells are presently unknown. ",
"section_name": "Notch and PlexinD1 signaling controls prostate cancer cell migration",
"section_num": null
},
{
"section_content": "Notch signaling has been associated with the activation of transcription factors involved in the so-called epithelial-mesenchymal transition (EMT), which promotes cell migration in development, as well as cancer invasion and metastasis [19,40,41,42,43].Thus, in order to identify the protein mediators downstream to PlexinD1, we analyzed the expression of EMT transcription factors Snail, Slug, Twist, Zeb1 and Zeb2 in PC3 prostate cancer cells.Interestingly, Slug levels were consistently upregulated upon expression of activated Notch1, or direct PlexinD1 overexpression ( The specific requirement for Sema3E/PlexinD1 signaling to regulate E-cadherin levels was further indicated by the functional rescue upon re-expression of a (non RNAi-sensitive) active form of the ligand p61-Sema3E (Fig 5M).Moreover, similar results were obtained by transfecting PlexinD1 cDNA in gene silenced cells (S8A Fig) .Interestingly, a point mutant of PlexinD1 known to lack GAP activity (RA-mutant; [4] was still fully competent to mediate E-cadherin downregulation (S8B Fig), consistent with the idea that PlexinD1 can mediate multiple signaling cascades in cancer cells [46].Furthermore, we found that E-cadherin upregulation in cancer cells subjected to PlexinD1 knock down was maintained upon transplantation in mice and tumor formation in vivo (S9A and S9B Fig). In further experiments we confirmed the relevant role of Slug to mediate Sema3E-induced E-cadherin downregulation (Fig 5N) and increased cancer cell migration (Fig 5O).Notably, it was previously reported that the nuclear translocation of another transcription factor (Snail) induced by Sema3E in ovarian cancer cells was mediated by the PI3K/AKT pathway [6].Thus we tested whether inhibitors of intracellular signaling effectors PI3K and MAPK could interfere with Sema3E/PlexinD1 dependent upregulation of Slug levels in prostate cancer cells.Intriguingly, while we could not detect any change in presence of 10μM LY294002 PI3K inhibitor (not shown), PlexinD1-dependent Slug upregulation (and consequent E-cadherin suppression) was blunted upon treatment with the MAPK inhibitor PD98059 (10μM) (Fig 5P ). Taken together, these data indicate that both Notch and PlexinD1 signaling can upregulate Slug expression in prostate cancer cells and concomitantly downregulate E-cadherin expression.In keeping with its role as transcriptional target of Notch, PlexinD1 could therefore also be envisaged as relevant effector of Notch signaling cascade. ",
"section_name": "Notch and PlexinD1 coordinately regulate gene expression in prostate cancer cells",
"section_num": null
},
{
"section_content": "As discussed above, transcriptional programs downstream to Notch signaling are known to foster cancer cell migration/invasion [47,48,49].Indeed, our data indicate that N1-ICD concomitantly promoted prostate cancer cells migration and downregulated E-cadherin levels.As this also correlated with increased PlexinD1 expression, we asked whether PlexinD1 could be the driver eliciting downstream regulatory mechanisms and cell migration.Thereby we transfected constitutively active Notch in cells carrying PlexinD1-targeted shRNAs; in this setting, PlexinD1 levels could not be induced by N1-ICD.Importantly, this prevented E-cadherin downregulation and a gain of cancer cell migration in response to constitutive Notch signaling (Fig 6A and6B).These data strongly suggest that PlexinD1 is required to mediate Notch activity in controlling cell migration. We also performed the complementary experiment: since Notch1 silencing caused reduced migration of PC3 and DU145 cells, correlating with lower PlexinD1 levels, we asked whether the forced expression of PlexinD1 could restore, at least in part, this functional loss.As shown in Fig 6C and6D, PlexinD1 overexpression was able to downregulate E-cadherin levels independent of Notch, and fully rescued migration defect (and E-cadherin loss) associated with reduced Notch1 activity, consistent with the idea that PlexinD1 is located downstream of Notch in the signaling cascade leading to E-cadherin regulation and increased cell migration.Importantly, this effect was also appreciable in in vivo setting, where PlexinD1 overexpression promoted metastatic cancer cell extravasation independently of Notch1 (Fig 6E and6F). ",
"section_name": "PlexinD1 acts downstream to Notch signaling to empower cancer cell migration",
"section_num": null
},
{
"section_content": "Previous studies have shown that Sema3E-PlexinD1 signaling is deregulated in human cancers; however, the molecular mechanisms responsible for PlexinD1 overexpression remained undetermined.According to our findings, canonical Notch signaling is a positive regulator of Plex-inD1 expression in an RBPjk dependent manner.This is supported by experimental evidence in a range of cells of different tumor origin, either subjected to Notch1 genetic silencing or treated with gamma secretase inhibitors to block this signaling cascade.Furthermore, we demonstrated the positive regulatory activity of Notch signaling on PLXND1 promoter, dependent on the canonical RBPJ-kappa pathway.Moreover, our data suggest that Notch1 and Notch3 may act redundantly in PlexinD1 regulation.Notably, Notch signaling is known to be upregulated during progression of colon, breast, ovarian and prostate cancers [16,50,51].Independently, it was shown that PlexinD1 is overexpressed in the same tumor types [5,6,7,8].Indeed, by TCGA data mining we found a positive correlation of NOTCH1 and NOTCH3 expression with PLXND1, supporting our hypothesis.In apparent conflict with this, a negative crosstalk has been observed in retinal vessel development where Notch signaling was found to downregulate PlexinD1 [11].However, PlexinD1 downregulation in zebrafish Notch signaling mutants has also been reported [52], which supports our data in human cells. The functional role of PlexinD1 signaling in cancer cell migration is under investigation.For instance, Sema3E revealed dual regulatory activity on migration.A recombinant molecule corresponding to the precursor full-length Sema3E may display inhibitory function [8,53].However, cancer cells often carry an autocrine loop of proteolytically processed isoform of Sema3E (dubbed p61-Sema3E) which instead promotes cancer cell migration in PlexinD1-dependent manner [5].Indeed, here we show that p61-Sema3E signaling enhanced prostate cancer cells migration, while knocking down the endogenous Sema3E or PlexinD1 levels inhibited it.Previous work has reported that PlexinD1 can functionally interact with ErbB2 and elicit invasion and metastasis [5].However, we found that this mechanism is unlikely to have a role in PC3 prostate cancer cells, as they express low levels of ErbB2 and the treatment with the kinase inhibitor lapatinib didn't seem to interfere with Sema3E/PlexinD1-mediated effects (M.R., unpublished results).In ovarian cancer cells it was reported that Sema3E signaling drives nuclear translocation of Snail, a transcription factor controlling E-cadherin expression.Our work now demonstrates that PlexinD1 regulates E-cadherin levels and cell migration in prostate cancer cells.Intriguingly, we could not detect any significant change of subcellular Snail localization upon N1-ICD or PlexinD1 overexpression in prostate cancer cells (data not shown), but we observed significant induction in the expression of Slug, another transcription factor known to drive E-cadherin loss.Notably, Slug was recently reported to be required for EMT and invasiveness of breast cancer cells in response to Notch signaling [49].Thus, based on our findings, we propose that in prostate cancer cells PlexinD1 mediates Slug upregulation elicited by Notch signaling.Interestingly, the molecular mechanisms linking Sema3E/PlexinD1 to Slug upregulation seem to be distinct from downstream pathways reported so far, and putatively implicate MAPK activity; further studies are warranted to elucidate this novel signaling cascade. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "In this study we have shown for the first time that Notch signaling (both Notch1 and Notch3 receptors) upregulates PlexinD1 expression at the promoter level in different cells.This regulatory mechanism is dependent on canonical Notch signaling, as RBPjk itself can induce Plex-inD1 expression, while this is blocked by its dominant negative form.Notch1 and Notch3 expression is positively correlated with that of PlexinD1 in patient cancer samples.We have revealed that PlexinD1 expression is required to mediate cancer cell migration induced by constitutive Notch signaling in prostate cancer cells; moreover, PlexinD1 expression is sufficient to promote cancer cell migration downstream of Notch.Notably, the overexpression of Notch1, Notch3 and PlexinD1 induces Slug transcription factor and downregulates E-cadherin levels in prostate cancer cells, a phenotype associated with increased cell migration and metastatic potential in vivo.This signaling cascade might be important in the gain of invasive and metastatic ability of human prostate tumors, and its therapeutic targeting could be envisaged by treatment with Notch signaling inhibitors. ",
"section_name": "Conclusion",
"section_num": null
}
] |
[
{
"section_content": "We gratefully acknowledge Silvia Giordano and Federico Bussolino (IRCC-Candiolo, Italy), Isabella Screpanti and Claudio Talora (Sapienza University of Rome, Italy), Takayasu Kato (University of Tsukuba, Japan), Alain Israel (Pasteur Institute, France) for generously sharing reagents.We thank Massimo Accardo for excellent technical assistance with experiments in mice and Guendalina Lombardo for help with IHC analysis.We thank all members of Tamagnone laboratory for discussions.The work was supported by grants from: Italian Association for Cancer Research (AIRC; IG #2014-15179), the Fondazione Piemontese per la Ricerca sul Cancro (FPRC-ONLUS; Grant \"MIUR 2010 Vaschetto-5 per mille 2010 MIUR\") and the Italian Ministry of Health (Ricerca Corrente 2016). ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "The work was supported by grants from Italian Association for Cancer Research (AIRC)(IG #2014-15179) and the Fondazione Piemontese per la Ricerca sul Cancro (FPRC-ONLUS)(Grant \"MIUR 2010 Vaschetto -5 per mille 2010 MIUR\"). ",
"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
}
] |
10.26508/lsa.202201663
|
Methionine uptake via the SLC43A2 transporter is essential for regulatory T-cell survival
|
<jats:p>Cell death, survival, or growth decisions in T-cell subsets depend on interplay between cytokine-dependent and metabolic processes. The metabolic requirements of T-regulatory cells (Tregs) for their survival and how these are satisfied remain unclear. Herein, we identified a necessary requirement of methionine uptake and usage for Tregs survival upon IL-2 deprivation. Activated Tregs have high methionine uptake and usage to S-adenosyl methionine, and this uptake is essential for Tregs survival in conditions of IL-2 deprivation. We identify a solute carrier protein SLC43A2 transporter, regulated in a Notch1-dependent manner that is necessary for this methionine uptake and Tregs viability. Collectively, we uncover a specifically regulated mechanism of methionine import in Tregs that is required for cells to adapt to cytokine withdrawal. We highlight the need for methionine availability and metabolism in contextually regulating cell death in this immunosuppressive population of T cells.</jats:p>
|
[
{
"section_content": "T cells play central roles in adaptive immune responses.To mount appropriate immune responses, T-cell subsets must adapt to a range of extracellular nutrient levels and environmental cues.Several signals push T cells out of quiescence and toward acquiring new functions.In this context, it is clear that metabolic reprogramming is central to the survival, differentiation, or functions of T cells and works in concert with key signaling systems and cytokines (Yang et al, 2013;Buck et al, 2015;He et al, 2017;Bantug et al, 2018;Chapman et al, 2020).What the different metabolic requirements are in distinct T-cell subtypes and how these are satisfied remain unclear.Depending upon the T-cell subset, distinct nutrients such as glucose, amino acids, or lipids control metabolic outputs that influence immune signaling and regulates the function and proliferation of T cells (Buck et al, 2015;Walls et al, 2016;Chapman et al, 2020).In general, T-cell activation in response to an antigen results in transcriptional and metabolic remodeling, leading to new functions which include the production of cytokines and molecules that support T-cell survival, expansion, and functional demands (Fox et al, 2005;MacIver et al, 2013;Blagih et al, 2015;Buck et al, 2015;Menk et al, 2018;Cho et al, 2019).Because T-cell subsets function in diverse and dynamic niches, they require different energetic and metabolic pathways for their survival and function (O'Neill et al, 2016;Scharping et al, 2016;Pan et al, 2017;Siska et al, 2017).For example, upon antigen activation, CD4 + T-effector cells switch from oxidative phosphorylation (OXPHOS) to glycolysis to meet metabolic demands, whereas antigenactivated CD4 + CD25 hi Foxp3 + T-regulatory cells (Tregs) switch to lipid oxidation and low glycolysis (Wang et al, 2011;He et al, 2017;Siska et al, 2017).Such differing uses of biosynthetic pathways distinguish T-cell fate choices.In this context, what the metabolic requirements that control Tregs survival are and how the cell satisfies them remain an important yet poorly explored area. Within this framework, multiple amino acids have unique roles in controlling T-cell function.Amino acids have diverse roles in metabolism and signaling and control multiple cellular programs.Studies in naïve T cells noted substantial changes in amino acid pools compared with activated T cells (Ananieva et al, 2014;Marchingo & Cantrell, 2022).Several amino acids such as leucine, glutamine, arginine, and tryptophan can regulate T-cell homeostasis and function, and the expression of distinct amino acid transporters is critical for these functions (Baban et al, 2009;Cobbold et al, 2009;Yan et al, 2010;Geiger et al, 2016;Ma et al, 2017).In T effectors (Teffs), critical roles for neutral and branched-chain amino acids were identified during T-cell expansion, for mTORC1 activation and for meeting requirements for bioenergetics (Sinclair et al, 2013(Sinclair et al, , 2019;;Ma et al, 2017).In Teffs, the solute carrier transporter SLC7A5 (which transports large neutral amino acids) was specifically up-regulated during activation, and this was required for their expansion in amino acid dependent contexts (Hayashi et al, 2013;Sinclair et al, 2013).CD4 + and CD8 + T cells deficient in SLC7A5 showed impaired clonal expansion and effector functions (Hayashi et al, 2013;Sinclair et al, 2013) and reduced mTORC1 activation concurrent with decreased glutamine and glucose uptake after T-cell activation (Sinclair et al, 2013).More recently, multifaceted roles for methionine were identified for T-cell activation and differentiation/expansion into Teffs (Sinclair et al, 2019).In the Teffs context, methionine-transport regulated by the SLC7A5 transporter was the rate-limiting, essential factor in the proliferation and differentiation of these T cells (Sinclair et al, 2019).Similarly, branched-chain amino acids transported by SLC3A2 and CD98 regulate activation and suppressor function in Tregs (Ikeda et al, 2017;Shi et al, 2019).These data suggest the likely existence of many as yet unknown roles of amino acids and amino acid transporters in regulating T-cell fates. Tregs are crucial for peripheral tolerance and immune homeostasis (Vignali et al, 2008;Corthay, 2009).Their immunosuppressive function is critical for pathologies such as autoimmunity, cancer, and tissue damage (Josefowicz et al, 2012;Ohkura et al, 2013).Tregs have unique metabolic requirements that are distinct from other T cells like the Teffs (Michalek et al, 2011;He et al, 2017;Yang et al, 2017).Tregs survive and protect themselves in dynamic and nutrient-limiting microenvironments, and these survival decisions have been best studied in the context of cytokine requirements in these cells.Therefore, understanding how Tregs regulate survival/ death programs becomes critical to more completely address how they function.One mechanism by which Tregs regulates effector CD4 + T-cell numbers is by depleting cytokines IL-2 in the microenvironment (Pandiyan et al, 2007;H öfer et al, 2012).Relevantly, activated CD4 + and CD8 + T effectors require cytokines like IL-2 for their survival and undergo apoptosis upon its withdrawal (Boyman et al, 2007;Purushothaman & Sarin, 2009).However, Tregs continue to survive in cultures without IL-2 for extended periods of time (Perumalsamy et al, 2012), and this indicates that additional, as yet unknown, factors enable the survival of Tregs.In this context, noncanonical (cytoplasmic) Notch1 activity can control Tregs survival, by regulating mitochondrial activity, and metabolism (Perumalsamy et al, 2012;Saini et al, 2022).Unlike in Teffs where Notch1 is localized in the nucleus, in Tregs Notch1 (which carries out protective effects) is predominantly cytoplasmic (Perumalsamy et al, 2012).Although these roles of Notch1 in enabling Tregs survival are critical, the unique metabolic requirements of Tregs for their survival and how these metabolic requirements are satisfied remain unknown. Herein, we report a unique amino acid requirement for Tregs survival when IL-2 is deprived.Upon IL-2 withdrawal, Tregs cells exhibit an increased requirement of a specific amino acid, methionine, which is critical for cell survival.This methionine uptake is enabled by the Notch1-mediated regulation of the activity of a specific solute carrier (SLC) transporter in Tregs.The transporter-mediated uptake and subsequent use of methionine is the limiting factor for Tregs survival.We thus identify an essential amino acid requirement for Tregs survival upon IL-2 withdrawal.This process is mediated by a novel Notch1-dependent amino acid transporter axis that regulates the sustained supply of methionine and thereby Tregs survival. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To investigate the status of amino acids in activated Tregs (referred to as Tregs in the text) survival contexts, we estimated changes in amino acid pools in murine Tregs upon IL-2 withdrawal.Relative steady-state concentrations of intracellular amino acids were quantitatively assessed over the first ~6 h after IL-2 withdrawal in Tregs, where they remained in complete medium with dialyzed serum (CMDS).Total metabolites were extracted from Tregs cultured in CMDS without IL-2 for 1, 3, and 6 h (Fig 1A : inset schematic), and amino acids were quantitatively assessed using targeted liquid chromatography/mass spectrometry (LC/MS/MS).Most of the amino acids did not show significant changes in their relative amounts (Fig 1A), indicating that Tregs continued to maintain an overall stable homeostasis with the external environment in this condition.Contrastingly, intracellular methionine amounts showed the most substantial decrease of all amino acids, decreasing as rapidly as within 1 h of cytokine withdrawal (Fig 1A).In addition, a decrease in the related sulfur amino acid cysteine, as well as histidine and tryptophan was also observed (Fig 1A).This suggested a possible shift in homeostasis in Tregs after IL-2 withdrawal, particularly toward increased methionine consumption.We therefore decided to subsequently focus on addressing a possible requirement specifically for methionine in Tregs post IL-2 withdrawal, based on the observation that methionine showed the most substantial decrease in amounts.In addition, we were intrigued by observations coming from a distinct context wherein methionine regulated CD4 + T cells activation, differentiation, and proliferation (Geltink & Pearce, 2019;Sinclair et al, 2019) and wondered how this context was distinct. Methionine is converted into its major metabolite SAM, and methyltransferases transfer the methyl group from SAM to produce a methylated substrate and SAH.We therefore next assessed the relative steady-state amounts of SAM and SAH, as part of the methionine cycle (Fig 1B).As observed with methionine levels shown earlier, from the same experiment, we observed that relative SAM amounts also significantly reduced in Tregs when cultured without IL-2 in complete medium (Fig 1B).As shown earlier, cysteine levels also showed a reduction (Fig 1B), consistent with expectations because cysteine and methionine are closely coupled to each other in the sulfur amino acid cycle.Together, these data suggest the possibility of an acute requirement of methionine in Tregs, upon IL-2 withdrawal, which is sustained by continuous uptake of this amino acid. The previous data provides a \"bulk estimate\" of steady-state levels of these metabolites.The experiment cannot unambiguously address if there is high uptake of extracellular methionine and/or its increased consumption in these Tregs post IL-2 withdrawal.To definitively address these possibilities, a separate \"flux\" experiment using a pulsing of stable-isotope 13 C5 15 N-labeled methionine was performed, using the experimental paradigm shown in Fig 1C .Activated Tregs were cultured in sulfur amino acid (SAA) dropout medium (DOSAA) (without IL-2 and with unlabeled methionine) for 1, 3, and 6 h.Twenty minutes before each of these time points, cells were washed with DOSAA, and to these cells, labeled methionine ( 13 C5 15 N) in DOSAA was added.After 20 min of labeled methionine addition, we collected cells and assessed the fraction of labeled versus unlabeled methionine in these cells (Fig 1D).Notably, we observed nearly complete replacement of unlabeled methionine with labeled methionine, with no detectable signal coming from the unlabeled methionine (which represents existing methionine pool (A) Amino acid changes in Tregs over time: Quantitative liquid chromatography-mass spectrometric (LC/MS/MS) analysis to determine relative changes in intracellular amino acid amounts in Tregs cultured in complete medium with dialyzed serum (CMDS) and without IL-2 for 1, 3, and 6 h.Amino acid amounts are plotted relative to T0 (onset of IL-2 withdrawal).Inset schematic: experimental timeline for assessment of metabolites in activated Tregs.(B) Methionine and sulfur cycle metabolites in Tregs: LC/MS/MS based analysis to determine relative changes in intracellular levels of methionine, SAM, SAH, and cysteine in Tregs cultured in CMDS without IL-2 for 1, 3, and 6 h.Data are plotted relative to T0 (also see Fig 1A for methionine and cysteine data).(C) Experimental approach to assess methionine uptake, and consumption in activated Tregs cultured in dropout of sulfur amino acids (DOSAA) and without IL-2.Tregs (in DOSAA+ unlabeled met) remained in culture until the specified time point.20 min before the specified time point (1, 3, or 6 h) cells were washed in DOSAA, and then DOSAA+ 13 C5 15 N-labeled methionine (150 μM) was added for 20 min, and cells were collected and processed for labeled metabolite analysis.(D) Schematic illustrating a stable-isotope label-based flux experiment for methionine uptake and methionine usage to SAM and SAH, at different time points after IL-2 withdrawal. 13C5 15 N methionine in DOSAA was pulsed for 20 min at the indicated time point (see Fig 1C).After extraction, the fraction of methionine with and without the 13 C5 15 N label was quantitatively estimated by targeted, quantitative LC/MS/MS, to indicate the extent of uptake of methionine.Similarly, the extent of label incorporation coming from methionine to SAM and SAH was assessed, to estimate the amount of methionine converted to SAM and SAM to SAH at that indicated time point.(E) Stable-isotope-based assessment of methionine uptake: after adding 13 C5 15 N labeled methionine at the indicated time point, the fraction of labeled to unlabeled methionine was estimated.Note: at all time points, ~100% of the methionine estimated was 13 C5 15 N labeled, indicating that all stores of (unlabeled) methionine was consumed and replaced within this time.Also see Fig S1 .(F) Stable-isotope-based assessment of methionine usage to SAM and SAH: for each time point indicated, after a 20-min addition of 13 C5 15 N-labeled methionine to Tregs, the amount of label incorporated into SAM and SAH was estimated and is indicated as the fraction labeled.A higher fraction labeled will indicate a greater consumption of methionine to SAM or SAH, in that period of time.S1).These data indicate that Tregs continuously take up external methionine and use it rapidly.Next, to unambiguously assess relative rates of methionine consumption and usage, we quantified the amount of labeled methionine that is converted into labeled SAM and subsequently labeled SAH, in the same experimental set up.Notably, labeled SAM amounts (to unlabeled SAM) were highest in 1 h after IL-2 withdrawal and steadily decreased over 3 and6 h (Fig 1F).Correspondingly, a similar trend was observed for SAH.These data indicate that in Tregs post IL-2 withdrawal, there is a high consumption of methionine to SAM (and SAM to SAH) and that the highest conversion of methionine to SAM occurs within this ~1 h period after IL-2 withdrawal. Collectively, these data demonstrate that Tregs (post IL-2 withdrawal) continuously take up extracellular methionine and consume this methionine to synthesize SAM, which itself is used (producing SAH).The highest methionine consumption occurs over the first ~1 h after IL-2 withdrawal. ",
"section_name": "Activated Tregs uptake and metabolize methionine upon cytokine withdrawal",
"section_num": null
},
{
"section_content": "Because activated Tregs survive after IL-2 deprivation (Perumalsamy et al, 2012), to investigate if methionine was required for this survival, Tregs were cultured without IL-2 either in SAA dropout or complete media with dialyzed serum for 24 h, and the extent of apoptotic damage was quantified.This was by using well established approaches to assess apoptosis in this exact experimental system, as shown earlier (Perumalsamy et al, 2009;Marcel & Sarin, 2016) Together, these data suggest that the protective role of SAAs in Tregs is specific to methionine.To further assess this methionine requirement for Tregs survival, in complementary experiments, we supplemented cells with ethionine, an antimetabolite and antagonist of methionine.Ethionine competitively blocks methionine uptake and use.The addition of ethionine reduced the methioninedependent cell survival after IL-2 withdrawal (Fig 2B).Finally, we better assessed the critical time window of methionine requirements for Tregs survival after IL-2 withdrawal.For this, we added back methionine at different time intervals (T0, T5, and T9) after IL-2 withdrawal and shifting cells to DOSAA.The protection of Tregs from apoptosis was restored to same extent as in Tregs cultured in CMDS where methionine was added back at the initial hours (i.e., T0 and T5) as compared with ~9 h after IL-2 withdrawal (T9) and shift to DOSAA (Fig 2C).As controls, we assessed the identity of Tregs generated from WT mice cultured with or without IL-2 either in SAA dropout or complete media, by immune staining for Foxp3 protein and found that Tregs in all these conditions retained Foxp3 expression (Fig 2D).Collectively, these data reveal a specific requirement of methionine uptake and usage for Tregs survival post IL-2 withdrawal. ",
"section_name": "Methionine is essential for the survival of Tregs in the absence of IL-2",
"section_num": null
},
{
"section_content": "In IL-2-limiting conditions, prior studies had established that the activity of nonnuclear Notch1 enables the survival of Tregs (Perumalsamy et al, 2012).We therefore asked if the methioninedependent survival of Tregs after IL-2 withdrawal also requires Notch1 activity.GSI-X (γ-secretase inhibitor-X), a pharmacological inhibitor of the enzyme γ-secretase was used to inhibit the cleavage and release of the Notch1 intracellular domain, as established in earlier studies (Perumalsamy et al, 2012;Saini et al, 2022).As a control, and consistent with earlier studies (Saini et al, 2022), Tregs cultured with GSI-X for 7 h showed a reduction in processed Notch1 protein and the canonical Notch1 transcriptional target Hes1 protein (Fig S3).Notably, the inhibition of Notch1 activity by GSI-X significantly reduced the protection conferred by methionine on Tregs survival (Fig 3A).It may be noted that to reduce any toxicity of GSI-X on Tregs, in these experiments, GSI-X and methionine were added 5 h after IL-2 withdrawal.Also note that in this experiment, the addition of methionine was not at the onset of IL-2 withdrawal as in earlier experiments.Therefore, just to ensure methionine-dependent protection of Tregs, we added a slightly higher concentration (200 μM) of methionine. We next asked if the methionine-dependent protection of Tregs apoptosis depended on Notch1.For this, we used Tregs isolated from Notch1 +/+ (Cre-ve) or Notch1 -/-(Cre+ve; Cd4-Cre::Notch1 lox/lox ) mice and activated in vitro.Notch1 -/-Tregs (unlike their Notch1 +/+ counterparts), undergo apoptosis when cultured without IL-2 for 24 h (Fig 3B).In this context, the addition of methionine failed to rescue the survival of .Contrastingly, and consistent with Fig 2A-C, the addition of methionine at the onset of IL-2 withdrawal protected activated Notch1 +/+ (Cre-ve) Tregs from apoptosis when cultured in the absence of SAAs (DOSAA) and IL-2 (Fig 3B).Notch1 +/+ (Cre-ve) Tregs survived well when cultured in CMDS without IL-2 as compared with Notch1 -/-(Cre+ve).Together, the data from these experiments show that Notch1 function is required for the methionine-mediated Tregs survival post IL-2 withdrawal.This also reaffirms earlier results (Perumalsamy et al, 2012) that demonstrate a requirement for Notch1 in Treg survival. To further understand if Notch1 had a role in mediating methionine uptake in Tregs, we assessed changes in the mRNA levels of several amino acid transporters after IL-2 withdrawal, both in the presence and absence of GSI-X (3 h treatment).These transporters belong to the solute carrier (SLC) superfamily, which are major transporters of several amino acids including methionine (Pizzagalli et al, 2021).Among the transporter transcripts analyzed, SLC6A17, SLC1A5, and SLC7A8 showed very high Ct values (>30) (Fig 3C), indicating very low/basal transcript levels in Tregs.In contrast, SLC3A2, SLC43A1, SLC43A2, and SLC7A5 all had higher mRNA expression as assessed by Ct values (Fig 3C).Interestingly, SLC43A2 and SLC43A1 transcript levels increased in Tregs cultured without IL-2 in complete medium for 3 h (Fig 3D).Notably, abrogating Notch1 activity by using GSI-X in Tregs cultured without IL-2 decreased the mRNA levels of only the SLC43A transporter (Fig 3D).None of the other SLC transporters revealed any trend of putative Notch1-dependent expression.In particular, the mRNA levels of SLC7A5, a transporter required for the import of methionine in CD4 + T cells (Sinclair et al, 2019), remained unaltered in Tregs.These data collectively suggested that the SLC43A transporters might have a role in the Notch1-mediated regulation of methionine uptake in Tregs. ",
"section_name": "Notch1 function is required for methionine uptake and survival of Tregs upon cytokine withdrawal",
"section_num": null
},
{
"section_content": "Of the transporter transcripts assessed, only the SLC43A family transporters had high transcript amounts, and SLC43A2 transcripts also showed a Notch1-dependent expression post IL-2 withdrawal.We, therefore, prioritized assessing possible roles of SLC43A2 and sought to understand if this transporter is required for Tregs survival upon IL-2 withdrawal, in the context of methionine availability.As an important comparison, we also assessed if SLC7A5, which regulates the import of methionine in CD4 + T cells (Sinclair et al, 2019), had any role to play in Tregs survival in the absence of IL-2.Using shRNA, we knocked down, respectively, SLC43A2 and SLC7A5 in Tregs (Fig 4A).The shRNAs were extensively validated for efficacy and specificity (see the Materials and Methods section and Table S1).We further confirmed that the knockdown of SLC43A2 did not affect SLC7A5 transcript levels for nonspecific reasons (Fig S4A).Using shRNAs, SLC43A2, and SLC7A5 were ablated in Tregs (protein levels shown in Fig S4B andC), and Tregs survival assessed after IL-2 withdrawal.The knockdown of SLC43A2, but not the knockdown of SLC7A5, specifically increased apoptotic damage as compared with Tregs transduced with scrambled control (Fig 4A).This suggests a necessary role for SLC43A2 in Tregs survival upon IL-2 withdrawal.We next asked whether the SLC43A2 transporter protein was required for the methionine-dependent survival of Tregs in IL-2-limiting conditions.Tregs were transduced with scrambled (control) and SLC43A2 shRNA (Fig S4D).The knockdown of SLC43A2 abrogated Tregs survival when cultured without IL-2 in CMDS and in methionine supplemented medium (Fig 4B).These data are in contrast to wild-type cells where methionine supplementation increased cell survival.SLC43A2 abrogated Tregs retained Foxp3 expression, as assessed by immune staining (Fig S4Ei andii), confirming that the knockdown of SLC43A2 does not alter Tregs identity as assessed based on this indicator.These data, therefore, show that the SLC43A2 transporter has a specific, necessary role in Tregs in enabling methionine-dependent survival in IL-2-limited medium. Because Notch1 could not protect Tregs in the absence of methionine, and the SLC43A2 mRNA levels appeared to be Notch1dependent (Fig 3D), we directly asked whether Notch1 regulates SLC43A2 protein levels in Tregs, and this thereby enables methionine-dependent survival.As important controls, we asked if SLC7A5, which is important for methionine import for the survival of CD4 + cells, had any Notch1 dependency in Tregs.For this, the levels of the SLC43A2 and SLC7A5 proteins in activated Notch1 +/+ and Notch1 -/-Tregs were determined.Notch1 -/-Tregs showed a significant reduction in SLC43A2 protein levels relative to Notch1 +/+ Tregs (Fig 4Ci).In contrast, no change was observed in the SLC7A5 transporter protein levels (Fig 4Cii).Furthermore, we also compared SLC43A2 protein abundance in Notch1 -/-Tregs as compared with Notch1 +/+ Tregs cultured without IL-2 for 4 h (Fig S5A).The SLC43A2 protein levels remain constitutively lower in the Notch1-null cells, consistent with a requirement for Notch1 to maintain SLC43A2 protein in Tregs.Notably, the protein levels of the SLC43A2 transporter or the SLC7A5 (which is important for methionine transport in CD4 + effector T-cells) transporter did not change in activated Notch1 +/+ (Cre-ve) and Notch1 -/-(Cre+ve) (Fig S5B).Consistent with these results, Notch1 inhibition by GSI also reduced SLC43A2 protein amounts but did not affect SLC7A5 protein levels in activated Tregs when compared with control (DMSO treated) cells (Fig S5Ci and ii).These data collectively indicated that the Notch1mediated regulation of SLC43A2 is specific to Tregs.Finally, freshly isolated Notch1 -/-Tregs showed decreased SLC43A2 protein levels, as compared with Notch1 +/+ Tregs (Fig 4D).This also further reiterates that the difference in the SLC43A2 protein level is not because of differential activation of Notch1 +/+ (Cre-ve) and Notch1 -/- (Cre+ve) Tregs.Note: the loss of Notch1 (Notch1-null) does not alter the expression/frequency of Tregs, but these cells show defects in suppressor function (see Marcel and Sarin [2016] and Saini et al [2022]).Together, these data suggest that amount of SLC43A2 transporter protein depends on Notch1 availability, and the loss of Notch1 (and not IL-2) reduces SLC43A2. As an additional control, we analyzed the SLC43A2 promoter for the putative RBP-jk-binding site consensus motif (CGTGGGAA) (Tun et al, 1994) in the promoter region, covering at least ~3,000 bp upstream of transcription initiation site.However, the SLC43A2 promoter region does not have any RBP-jk-binding site.This hinted at possible noncanonical or indirect Notch1 function regulating SLC43A2 amounts.Separately, in these exact conditions, earlier studies had established that the Notch1 intracellular domain (NIC) activity from the cytoplasm protects Tregs from apoptosis in IL-2-limiting conditions (Perumalsamy et al, 2012).This was established by transducing Notch1 -/-Tregs with recombinant NIC tagged to a nuclear export signal to enrich localization of NIC in the cytoplasm of Tregs (Shin et al, 2006;Perumalsamy et al, 2012).We, therefore, examined the effect of NIC-NES on SLC43A2 protein levels, to determine if similar noncanonical Notch1 functionality is required for the SLC43A2 function in this methionine uptake context in Tregs.The overexpression of NIC-NES by retroviral transduction in Tregs (where Notch1 is highly enriched in the cytoplasm [Perumalsamy et al, 2012]) specifically increased protein levels of SLC43A2 in Notch1 -/-Tregs (Fig 4E).Finally, we asked if the overexpression of SLC43A2 in Notch1 -/-Tregs was in itself sufficient to protect these cells from apoptosis triggered by IL-2 withdrawal and if Notch1 had additional roles in enabling Tregs survival.Overexpressing SLC43A2 only partially rescued Notch1 -/-Tregs from apoptosis (Fig 4F).Note: for activated Notch1 -/-Tregs transduced with SLC43A2 and NIC-NES and pBABE vector control, the overexpression of NIC-NES and SLC43A2 in Notch1 -/-was verified (Fig S5D).The rescue by overexpressing SLC43A2 was not equivalent to that provided by overexpression of NIC-NES (Fig 4F ), suggesting additional factors in the survival program that are under Notch1 regulation.Collectively, these data show that SLC43A2 is required for the methionine-dependent survival of Tregs in IL-2 deficient conditions, in a Notch1-dependent manner. ",
"section_name": "SLC43A2 transporter is essential for methionine-dependent Tregs survival",
"section_num": null
},
{
"section_content": "Understanding processes that enable death-survival decisions in T cells is a prerequisite to decipher how T cells regulate adaptive immune responses.Based on our data, we present a simple model for a necessary requirement for Tregs survival after IL-2 withdrawal (Fig 4G).Upon IL-2 withdrawal, Tregs require the sustained uptake, transport, and usage of methionine.To enable this, Notch1 (nonnuclear) functions to regulate the expression of a specific solute carrier transporter, SLC43A2.SLC43A2 allows Tregs to take up methionine, which is subsequently used (Fig 4G).A reduction in SLC43A2 protein because of reduced Notch1 activity in these contexts abolishes Tregs survival conferred by methionine.At this stage, these data suggest a coordinated \"top-down\" (Shyer et al, 2020) metabolic survival signaling and metabolic cascade in Tregs, where the IL-2 withdrawal and the activity of Notch1 together coordinate a methionine-dependent survival program. To understand the full metabolic programs that regulate cell death/survival/growth programs, it is useful to separately question what metabolites are contextually needed and how these metabolic needs are sustained, versus what are the roles of specific metabolic programs in cells.In this study, we have only identified methionine uptake and usage as a limiting step in Tregs survival upon IL-2 withdrawal, with the SLC43A2 transporter sustaining this supply of sufficient methionine.This study, therefore, addresses a critical metabolic requirement needed for Tregs survival and how this requirement is satisfied.The SLC class of transporters constitutes a large superfamily with increasingly important metabolic roles (Schumann et al, 2020;Song et al, 2020), of which the more selective amino acid transporters (such as SLC7 or SLC43A) remain poorly studied.Currently, the critical roles of SLC class transporters for other metabolites, particularly glucose import via GLUT or SLC2A transporters in controlling T-cell metabolism to enable differentiation or activation, are better known (Jacobs et al, 2008;MacIver et al, 2008;Basu et al, 2015;Gerriets et al, 2015).As gatekeepers of nutrient (including amino acid) availability and therefore enablers of metabolic programs, it is likely that the amino acid transporting SLC proteins will also play roles in the survival or development of variety of cells and likely also cross-talk with the glucose-dependent responses.Understanding how these transporters function in different contexts and how they are regulated by cytokine-dependent and nutrient signaling systems will be necessary to further understand how unique metabolic programs are enabled in T cells.Separately, it will be important to understand how T-cell fate-regulating signaling systems such as mTORC1, Akt, or Notch control the activity or functions of these transporters, to sustain specific metabolic programs that determine activation, differentiation, or survival/death decisions in lymphocytes.For example, in other cellular contexts, earlier studies have suggested that this form of NIC-mediated Notch1 function can converge on mTORC1 function to enable cell survival (Perumalsamy et al, 2012).In all likelihood, there will be contextual differences in the specific mechanisms of apoptosis protection mediated by methionine.All of these form exciting future directions of research. Finally, the various possible functions of methionine in enabling Tregs survival remain unknown.Methionine, primarily through its metabolic product SAM, controls a variety of fundamental metabolic, signaling and regulatory processes in various cells (Walvekar et al, 2018b;Sanderson et al, 2019;Walvekar & Laxman, 2019;Lio & Huang, 2020;Roy et al, 2020).The presence or absence of methionine itself triggers cascading, hierarchically organized metabolic programs in cells (including glucose and other amino acid metabolism) (Walvekar et al, 2018b;Roy et al, 2020), to alter proliferation versus survival trajectories.In the context of T cells, in a relevant example of T effectors, antigenic stimulation-induced methionine uptake and usage appears to drive a program of nucleotides and proteins methylation for multiple (and not a single) fundamental processes (Sinclair et al, 2019).This is a contrasting context where cell growth and proliferation predominates (Sinclair et al, 2013;Geltink & Pearce, 2019), in contrast to the present study where for Tregs survival (where there is no growth or differentiation), cells require substantial methionine uptake and consumption, to presumably enable a distinct survival program.Reports in T helper (T h ) cells find that methionine metabolism maintains SAM pools to shape cell proliferation in part via altering histone modifications (Roy et al, 2020).It is therefore apparent that the contextual use of methionine in these different T-cell populations can have important yet poorly identified roles in regulating various functions.Therefore, understanding the methionine-dependent metabolic programs in Tregs and how they regulate signaling and regulatory outputs to regulate Tregs functions for adaptive immune responses will be an exciting direction of future enquiry. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Mice C57BL/6J mice were procured from the Jackson Laboratory.The Notch1 lox/lox and Cd4-Cre::Notch1 lox/lox (Notch1 -/-) mouse strains were a kind gift from F Radtke ( École Polytechnique Federale de Lausanne [EPFL]) (Wolfer et al, 2001).The Institutional Animal Ethics Committees of the National Centre for Biological Sciences (NCBS) and the Institute for Stem Cell Science and Regenerative Medicine (inStem), Bangalore, India, approved procedures involving mice, as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India.Breeding colonies were maintained in-house (NCBS/inStem Animal Care and Resource Center) in controlled environments and in high-barrier specific pathogen-free conditions.Colonies were regularly tested for the global pathogen panel (based on FELASA recommendations). ",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "Tregs and Teffs were isolated from spleens of 8-12-wk-old mice of mixed gender as described previously (Saini et al, 2022).CD4 + CD25 + Tregs and CD4 + CD25 -Teffs were isolated using the Dynabeads FlowComp Mouse CD4 + CD25 + Treg cells Kit (11463D; Invitrogen) following the manufacturer's instructions and as previously described (Perumalsamy et al, 2012).Approximately 120-150 × 10 6 lymphocytes cells were used to isolate ~2 × 10 6 Tregs and ~12 × 10 6 Teffs per column.Tregs and Teffs were activated at a density of ~2 × 10 6 /ml with magnetic beads (Invitrogen) coated with CD3 and CD28 antibodies (20 μl/ml) in a 24-well plate.Note: no IL-2 or TGF-β was added during the activation process.After 38-44 h, beads were removed by magnetic separation, and activated Tregs were washed and used for experiments.Cells were cultured in RPMI 1640 (Thermo Fisher Scientific) supplemented with 0.1% penicillin/ streptomycin, glutamine, and 5% heat-inactivated fetal bovine serum (Complete Medium, CM) or 5% heat-inactivated fetal bovine dialyzed serum (CMDS) whenever required.When required, T cells were cultured in RPMI without L-cysteine, L-cystine, L-methionine (MP Biomedicals) supplemented with 0.1% penicillin/streptomycin, glutamine and 5% heat-inactivated fetal bovine dialyzed serum (DOSAA). ",
"section_name": "T-cell subsets isolation and activation",
"section_num": null
},
{
"section_content": "The HEK293T (HEK) and NIH3T3 cell lines were obtained from the American Type Culture Collection (ATCC).Cell lines were cultured in DMEM (Thermo Fisher Scientific) supplemented with 0.1% penicillin/ streptomycin, glutamine and 10% heat-inactivated fetal bovine serum (Scientific Hyclone TM).HEK cells were used for retroviral packaging till passage 20.Cells were maintained at 37°C with 5% CO 2 .The cells were routinely tested free from mycoplasma by using the MycoAlert Kit (Lonza). ",
"section_name": "Cell lines",
"section_num": null
},
{
"section_content": "",
"section_name": "Reagents and antibodies",
"section_num": null
},
{
"section_content": "TRIzol (15596026) and SYBR Green Master Mix (Thermo Fisher Scientific); PrimeScript first strand cDNA Synthesis Kit (6110A) (Takara Bio); Recombinant IL-2 (R&D Systems); magnetic beads coated with anti-CD3 and anti-CD28 antibodies (Invitrogen); γ-Secretase Inhibitor-X (GSI-X, 565771) and puromycin (508838) (Calbiochem-Merck Millipore); X-treme-GENE (6366236001), histopaque (10831), L-ethionine (E1260), L-cysteine (168149), and L-methionine (M9625) (Sigma-Aldrich); 13C515N-labeled L-methionine (CNLM-759-H-0.1)(Cambridge Isotope Laboratories, Inc.).All other reagents were purchased from Himedia or Sigma-Aldrich.L-Ethionine, L-cysteine, and L-methionine stocks were made in autoclaved distilled water, filter sterilized, and stored at -80°C. 1 mM GSI-X was made in dimethylsulfoxide and stored at -20°C.Fresh aliquots were used for each experiment.Antibodies PA5-71365 against SLC7A5 (Life Technologies/Thermo Fisher Scientific); 11-5773-80 against Foxp3 (eBiosciences/Thermo Fisher Scientific); ab186444 against SLC43A2 and mN1A (128076) against Notch1 (Abcam); AB15470 against Hes-1 (Millipore Sigma), ACTN05, MS-1295-P to actin and MS-581-P0 to tubulin (Neomarker); 11-4321-80 isotype control (eBiosciences/Thermo Fisher Scientific); horseradish peroxidase-linked anti-mouse (7076S) and anti-rabbit IgG (7074P2) (Cell Signaling Technology). ",
"section_name": "Reagents",
"section_num": null
},
{
"section_content": "Verified murine shRNA plasmids to SLC43A2 (TR5143399), SLC7A5 (TG513945), scrambled control shRNA (TR30012), and pVMV6 entry-SLC43A2 Human Tagged ORF Clone (RC206639) were from Origene.The set of four shRNA constructs supplied were screened individually for knockdown of protein expression in NIH3T3 (ATCC) cells.The construct[s] (usually one or two) that effectively knocked down the target protein was used in the retroviral transductions of T-cells.The target shRNA sequence used in the study is provided in Table S1. The pBABE and pBABE-NIC-NES plasmids were gifts from BA Osborne (University of Massachusetts/Amherst).The Human SLC43A2 gene from pCMV6 entry vector was subcloned into the pBABE vector.Primers used for subcloning are listed in Table S1.Construct sequences were verified by automated Sanger sequencing conducted in-house. ",
"section_name": "Plasmids",
"section_num": null
},
{
"section_content": "HEKs (0.25 × 10 6 ) were seeded in 35-mm dishes (Greiner Bio-one).Cells (50-60% confluent) were transfected the next day with retroviruses containing the gene of interest (1.5 μg) and packaging vector pCL-Eco (1.5 μg for plasmids and 2 μg for shRNA) using X-tremeGENE.Retrovirus transduction was as described earlier (Saini et al, 2022).Briefly, the virus in the supernatant was concentrated by centrifugation at 21,000g for 1 h 30 min at 4°C.Tregs stimulated with anti-CD3 -CD28-coated beads for 24 h were infected using a cocktail of the concentrated virus, RPMI-CM, 10 mM Hepes, and 8 μg/ml sequa-brene by centrifugation at 600g for 90 min at 25°C.Twenty-four hours postinfection, Tregs were harvested, beads removed by magnetic separation, and continued in culture (0.5 × 10 6 cells/ml) in RPMI-CM supplemented with IL-2 (1 μg/ml) to minimize stress to the cells.After 18-24 h, puromycin (1 μg/ml) was added to the media along with IL-2 (1 μg/ml) to enrich transfected cells.48 h after the addition of puromycin, live cells were selected over histopaque (1.083 g/ml density) by density-gradient centrifugation at 300g for 20 min.Cells (~0.6-0.8 × 10 6 ) were washed twice in RPMI-CM and PBS and cultured in media supplemented with IL-2 (1 μg/ ml) and IL-7 (2 ng/ml) for another 22-24 h and used for apoptosis assays.Knockdown or overexpression of targeted genes was assessed by Western blot analysis. ",
"section_name": "Retroviral transductions",
"section_num": null
},
{
"section_content": "Cells (0.4-0.6 × 10 6 ) were lysed in 25 μl SDS lysis buffer, and cell lysates were resolved by 10% SDS-polyacrylamide gel electrophoresis followed by Western blot analysis as described earlier (Saini et al, 2022).Primary antibodies were used at the following dilution: Notch1 (1:500), SLC43A2 (1:500), SLC7A5 (1:500), actin (1: 1,000), HES1 (1:500), and tubulin (1:1,000) diluted in 5% skimmed milk in Tris-buffered saline-Tween 20 (TBST).Horseradish peroxidaseconjugated secondary antibody was used at a 1:1,000 dilution.The membranes were developed using Super Signal West Dura substrate (Thermo Fisher Scientific), and images were acquired in iBright FL1000 (Invitrogen). ",
"section_name": "Western Blot analysis",
"section_num": null
},
{
"section_content": "Tregs (2 × 10 6 , activated for 38-40 h) were cultured in CMDS without IL-2 for 1, 3, and 6 h.After the incubation, the cells were pelleted down at 600g for 3 min, and metabolites extracted.Briefly, 1 ml of ice-cold 10% methanol was added without disturbing the pellet (to quench metabolism) and further centrifuged at 600g for 3 min at 4°C.Furthermore, 1 ml of 80% methanol (maintained at -45°C) was added to the pellet, vortexed for 15 s, and incubated at -45°C for 15 min for metabolite extraction.The tubes were vortexed (15 s) and centrifuged at 21,000g for 10 min at -5°C.The supernatant (900 μl) was transferred into fresh tubes, recentrifuged at 21,000g for 10 min at -5°C, and the supernatant was removed and dried using a SpeedVac.The samples were stored at -80°C briefly, before analysis by targeted LC/MS/MS to assess the specified metabolite amounts, using methods described earlier (Walvekar et al, 2018a).For methionine uptake experiments, Tregs (2 × 10 6 , activated for 38-40 h) were harvested and washed three times with PBS and further cultured in DOSAA and unlabeled methionine (150 μM) in the absence of IL-2 for 1, 3, and 6 h.20 min before harvesting and metabolite extraction, the cells were harvested, washed once with DOSAA, and cultured with 13 C5 15 N-labeled methionine (150 μM) without IL-2.Subsequently, the relative amounts of labeled methionine (see Q1/Q3 details) and label incorporation from methionine to SAM or SAH were measured using targeted LC/MS/MS based approaches.MS-Q1/Q3 (Parent/Product) parameters used: methionine (Q1/Q3 150.1/56),SAM (Q1/Q3 399/250), SAH (Q1/Q3 385/136), 13 C5 15 N Methionine (5 13 C, 1 15 N Q1/Q3 155.1/60), 13 C5 15 N SAM (5 13 C Q1/Q3 405/250), and 13 C5 15 N SAH (4 13 C Q1/Q3 390/136).Mass spectrometer used: AB Sciex 6500 QTRAP. ",
"section_name": "Metabolic profiling",
"section_num": null
},
{
"section_content": "Relative metabolite amounts are typically shown.For this, the first time point (T0) data is normalized to 1, and subsequent samples were compared relative to T0. Raw and normalized data are shown in worksheet 1. ",
"section_name": "Data normalization",
"section_num": null
},
{
"section_content": "Tregs (4 × 10 6 , activated for 38-40 h) were harvested and cultured without IL-2 in the presence and absence of GSI-X (10 μM) for 3 h.After the incubation, cells were lysed in 700 μl of TRIzol (Thermo Fisher Scientific).RNA was isolated according to the manufacturer's instructions.cDNA was synthesized with 1 μg RNA using the Pri-meScript first strand cDNA Synthesis Kit (Takara Bio).cDNA was diluted in a 1:5 ratio and used for real-time PCR using the Maxima SYBR Green qPCR Master Mix and Bio-Rad CFX96 Touch Real-Time PCR Detection System.Relative change in transcript levels was calculated using the 2 -ΔΔCt method (Livak & Schmittgen, 2001).Hypoxanthine-guanine phosphoribosyltransferase (HPRT) or actin was used as the reference gene.All primers used are listed in Table S1. ",
"section_name": "Real-time PCR analysis",
"section_num": null
},
{
"section_content": "Tregs were washed with PBS thrice and cultured at 0.3 × 10 6 cells/ml 48-well plates in the absence of IL-2 and with the required treatment for 18-22 h.At the end of the treatment, cells were harvested and stained with Hoechst 33342 (1 μg/ml in PBS) in the dark at ambient temperature for 5 min.Cells were washed to remove excess dye, and pellets resuspended in 20 μl PBS and imaged to score nuclear morphology using a fluorescent microscope (Olympus BX-60) (Perumalsamy et al, 2012).Samples were counted double blind, and ~200 cells across several fields were scored for nuclei with normal or apoptotic morphology.Field views showing live and dead cells were acquired using inverted Olympus IX-73, 60× Objective, NA 0.70.Images were processed using Fijji-Image J. ",
"section_name": "Apoptosis assay",
"section_num": null
},
{
"section_content": "0.3 × 10 6 Tregs were adhered onto cut confocal dishes coated with 1 mg/ml poly-D-lysine in PBS for 10 min and fixed with 2% freshly reconstituted paraformaldehyde for 20 min in the dark at room temperature.After fixation, Tregs were permeabilized using 0.2% NP-40 in PBS for 10 min at room temperature and blocked with 5% BSA in PBS for 1 h at room temperature.Cells were incubated with a Foxp3 antibody tagged with FITC (1:100) or isotype control antibody diluted in 5% BSA in PBS overnight at 4°C.Cells were washed twice with PBS and stained with Hoechst 33342 (1 μg/ml) for 10 min and 10-15 random fields were imaged using Olympus FV3000 using Plan-Apochromat 63X NA 1.35 oil-immersion objective.Images were processed to remove background based on Isotype controls. ",
"section_name": "Immunofluorescence analysis",
"section_num": null
},
{
"section_content": "Data are represented as mean ± SD of two or three independent experiments (as indicated).Statistical significance was calculated using the two-tailed t test, and P-values ≤ 0.05 were considered as significance.Western blots were analyzed using ImageJ software and processed with Adobe Photoshop.Graphs and heat maps were prepared using GraphPad Prism, and figures were prepared using Adobe Illustrator. ",
"section_name": "Statistical analysis and data presentation",
"section_num": null
}
] |
[
{
"section_content": "We acknowledge Freddy Radtke, EPFL, Switzerland, for the Notch1 lox/lox and Cd4-Cre::Notch1 lox/lox mice; Barbara A Osborne, Amherst, USA, for pBABE and pBABE-NIC-NES plasmids; and I Verma (plasmid #12371; Addgene) for the pCL-Eco construct.We acknowledge Bangalore Life Science Cluster (BLiSC), the Mass Spectrometry Facility, and Animal Care and Resource Center (ACRC) of NCBS-TIFR and DBT-inStem, Bangalore, India.We especially thank Sreesa Sreedharan for help in completing some metabolite analysis experiments.A Sarin and S Laxman acknowledge support from the Department of Biotechnology (DBT), Government of India grant BT/PR13446/COE/34/30/2015. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "The authors confirm that the data supporting the findings of this study are available within the article (and/or) its supplementary materials. ",
"section_name": "Data Availability",
"section_num": null
},
{
"section_content": "Supplementary information is available at https://doi.org/10.26508/lsa.202201663. N Saini: data curation, formal analysis, validation, investigation, visualization, methodology, and writing-original draft, review, and editing.A Naaz: data curation, formal analysis, validation, investigation, visualization, methodology, and writing-original draft, review, and editing.SP Metur: data curation, formal analysis, validation, investigation, and methodology.P Gahlot: data curation, formal analysis, validation, investigation, and methodology.A Walvekar: data curation, formal analysis, validation, investigation, and methodology.A Dutta: data curation, formal analysis, and investigation.U Davathamizhan: data curation, formal analysis, and investigation.A Sarin: conceptualization, resources, supervision, funding acquisition, visualization, methodology, project administration, and writing-original draft, review, and editing.S Laxman: conceptualization, resources, supervision, funding acquisition, visualization, methodology, project administration, and writing-original draft, review, and editing. The authors declare that they have no conflict of interest. ",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "Supplementary information is available at https://doi.org/10.26508/lsa.202201663. ",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "N Saini: data curation, formal analysis, validation, investigation, visualization, methodology, and writing-original draft, review, and editing.A Naaz: data curation, formal analysis, validation, investigation, visualization, methodology, and writing-original draft, review, and editing.SP Metur: data curation, formal analysis, validation, investigation, and methodology.P Gahlot: data curation, formal analysis, validation, investigation, and methodology.A Walvekar: data curation, formal analysis, validation, investigation, and methodology.A Dutta: data curation, formal analysis, and investigation.U Davathamizhan: data curation, formal analysis, and investigation.A Sarin: conceptualization, resources, supervision, funding acquisition, visualization, methodology, project administration, and writing-original draft, review, and editing.S Laxman: conceptualization, resources, supervision, funding acquisition, visualization, methodology, project administration, and writing-original draft, review, and editing. ",
"section_name": "Author Contributions",
"section_num": null
},
{
"section_content": "The authors declare that they have no conflict of interest. ",
"section_name": "Conflict of Interest Statement",
"section_num": null
}
] |
10.1038/s41591-022-01927-8
|
Detection of early seeding of Richter transformation in chronic lymphocytic leukemia
|
<jats:title>Abstract</jats:title><jats:p>Richter transformation (RT) is a paradigmatic evolution of chronic lymphocytic leukemia (CLL) into a very aggressive large B cell lymphoma conferring a dismal prognosis. The mechanisms driving RT remain largely unknown. We characterized the whole genome, epigenome and transcriptome, combined with single-cell DNA/RNA-sequencing analyses and functional experiments, of 19 cases of CLL developing RT. Studying 54 longitudinal samples covering up to 19 years of disease course, we uncovered minute subclones carrying genomic, immunogenetic and transcriptomic features of RT cells already at CLL diagnosis, which were dormant for up to 19 years before transformation. We also identified new driver alterations, discovered a new mutational signature (SBS-RT), recognized an oxidative phosphorylation (OXPHOS)<jats:sup>high</jats:sup>–B cell receptor (BCR)<jats:sup>low</jats:sup>-signaling transcriptional axis in RT and showed that OXPHOS inhibition reduces the proliferation of RT cells. These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover potential therapeutic targets for RT.</jats:p>
|
[
{
"section_content": "",
"section_name": "Articles",
"section_num": null
},
{
"section_content": "plasmablastic lymphoma (RT-PBL; n = 1) or prolymphocytic leukemia (RT-PLL; n = 1).Nontumor samples were available in 12 patients.RT occurred simultaneously with CLL at diagnosis (n = 3) or after up to 19 years following different lines of treatment with CIT (n = 6) and targeted therapies (n = 10; BCR inhibitors, ibrutinib n = 6; duvelisib n = 2; idelalisib n = 1; and BCL2 inhibitor, venetoclax n = 1).All instances of RT were clonally related to CLL, 15 tumors had unmutated IGHV (U-CLL) and 4 had mutated IGHV (M-CLL).Whole-genome sequencing (WGS) data were integrated with bulk epigenetic and transcriptomic analyses as well as single-cell DNA and RNA sequencing (Fig. 1a, Extended Data Fig. 1 and Supplementary Tables 1 and2). The WGS and epigenome of CLL and RT revealed a concordant increased complexity from CLL diagnosis to relapse and RT (Fig. 1b, Extended Data Fig. 2a and Supplementary Tables 345678).The RT genomes carried a median of 1.8 mutations per megabase, 18 copy number alterations (CNAs) and 37 structural variants (SVs) that contrasted with 1.1 mutations per megabase, 4 CNAs and 5 SVs observed at CLL diagnosis.No major differences were seen among RT occurring after different therapies (Fig. 1b and Extended Data Fig. 2b).We discovered new driver genes and mechanisms in RT, expanding previous observations [12][13][14][15][16][17][18][21][22][23][24] (Fig. 1c, Extended Data Fig. 2c-e, Supplementary Fig. 1 and Supplementary Tables 9 and10). The min alterations involved cell-cycle regulators (17 of 19, 89%), chromatin modifiers (79%), MYC (74%), nuclear factor (NF)-κB (74%) and NOTCH (32%) pathways.These aberrations were simultaneously present in most cases but alterations in MYC and NOTCH pathways only co-occurred in 2 of 19 cases (Fig. 1c).Aberrations in genes such as TP53, NOTCH1, BIRC3, EGR2 and NFKBIE were usually present and clonally dominant after the first CLL sample, whereas others were only detected at RT or during the disease course (for example CDKN2A/B, CDKN1A/B, ARID1A, CREBBP, TRAF3 and TNFAIP3) (Fig. 1c).New alterations included deletions of CDKN1A and CDKN1B in five cases of RT associated with downregulation of their expression, one immunoglobulin (IG)-CDK6 translocation and one CCND2 mutation already present at CLL diagnosis, and CCND3-IG and MYCN-IG translocations acquired at RT in two different cases (Fig. 1d,e, Extended Data Fig. 3a,b and Supplementary Table 11).Most chromatin remodelers were affected by deletions with reduced gene expression.New alterations in this group were deletions of ARID4B and truncations of CREBBP 25 and SMARCA4 (ref.16 ) by translocations and chromoplexy (Fig. 1f and Extended Data Fig. 3c-e).We also identified recurrent IRF4 alterations in RT, which have been linked to increased MYC levels in CLL 26 .BTK/PLCG2 or BCL2 mutations were not detected in any RT after treatment with BCR or BCL2 inhibitors, respectively.Notably, the two cases of M-CLL developing RT after targeted therapies carried the IGLV3-21 R110 mutation, which triggers cell-autonomous BCR signaling 27 (Fig. 1c). In addition to the high frequency of CNAs previously identified in RT 11,14 , we observed a high number of complex structural alterations (Fig. 1c).Chromothripsis was found in eight RT tumors targeting CDKN2A/B and the new CDKN1B in five and one cases, respectively, and MYC, MGA, SPEN, TNFAIP3 and chromatin remodeling genes in additional cases (Fig. 1g and Extended Data Fig. 3f-j). Altogether, our analyses expand the catalog of driver genes, pathways and mechanisms involved in RT and recognize a similar distribution of these alterations in RT after different therapies, suggesting that treatment-specific pressure is not a major determinant of the driver genomic landscape of these tumors. ",
"section_name": "NATuRE MEDiCiNE",
"section_num": null
},
{
"section_content": "To understand the increased mutational burden of RT, we explored the mutational processes re-shaping the genome of CLL and RT.An unsupervised analysis showed that the mutational profile of RT was notably different from M-CLL and U-CLL before therapy (ICGC-CLL cohort, n = 147) 28 or at post-treatment relapse (independent cohort of 27 CLL post-treatment samples) (Fig. 2a).We identified 11 mutational signatures distributed genome-wide and 2 in clustered mutations (Extended Data Fig. 4 and Supplementary Tables 121314).Among the former, we extracted a new signature characterized by (T>A)A and, to a lesser extent, (T>C/G)A mutations not recognized previously in any cancer type, including CLL and DLBCL [28][29][30][31][32][33] .We named this single-base substitution signature, SBS-RT (Fig. 2b).SBS-RT was present in the RT sample of 7 of 18 patients, 1 of 6 after CIT and 6 of 10 after multiple therapies, including targeted agents and detected in all subtypes of transformation (RT-DLBCL, RT-PBL and RT-PLL) (Fig. 2c and Supplementary Table 15).It was also present in CLL samples before RT in patients 12 and 3,299 but was not identified in the reanalysis of our ICGC-CLL or post-treatment CLL cohorts.None of the patients in these two additional cohorts had evidence of RT (median follow-up 9.8 years, range 0.2-30.4)(Fig. 2c, Extended Data Fig. 5a and Supplementary Table 15).Further characterization of this new signature showed (1) a modest correlation between SBS-RT and total number of mutations (R = 0.79, P = 0.11); (2) SBS-RT mutations present in all different chromatin states and early/late replicating regions although with a moderate enrichment in heterochromatin/late replication; and (3) lack of replication and transcriptional strand bias (Extended Data Fig. 5b-f and Supplementary Table 16). Among the remaining ten genome-wide signatures, five were previously identified in CLL and DLBCL (SBS1 and SBS5 (clock-like), SBS8 (unknown etiology), SBS9 (attributed to polymerase eta) and SBS18 (possibly damage by reactive oxygen species)); three had been only found in DLBCL (SBS2 and SBS13 (APOBEC enzymes) and SBS17b (unknown)); and two have been recently described related to treatments with melphalan 34 or ganciclovir 35 , which were named here as SBS-melphalan and SBS-ganciclovir, respectively (Fig. 2b,c and Extended Data Fig. 4).SBS-melphalan was found in three RT cases, two had received melphalan as a conditioning of their allogenic stem-cell transplant 1.9 and 4.2 years before RT, respectively.SBS-ganciclovir was found in the RT sample of one patient that had received valganciclovir (prodrug of ganciclovir) due to cytomegalovirus reactivation (Fig. 2c,d and Extended Data Fig. 1a).Notably, all cases with the new SBS-RT at time of RT had been treated with the alkylating agents bendamustine (n = 5) or chlorambucil (n = 2) during their CLL history at a median of 2.9 years (range 0.7 to 6.8) before RT.Contrarily, RT cases lacking the SBS-RT had never received these drugs (Fig. 2c,d and Extended Data Fig. 1a). To time the activity of each mutational process, we reconstructed the phylogenetic tree for the 11 patients with multiple synchronous (n = 2) or longitudinal (n = 9) samples and germline available and measured the contribution of each signature to the mutational profile of each subclone.The major subclone at time of transformation was named 'RT subclone' (Supplementary Table 17).As expected, clock-like mutational signatures were present all along the phylogeny (constantly acquired), whereas SBS9 was found only in the trunk of the two M-CLL tumors (patients 365 and 19; early events).DLBCL-related signatures, SBS-ganciclovir, SBS-melphalan and SBS-RT were found in single RT subclones in six cases while two cases carried two simultaneous subclones with SBS-RT (patients 12 and 19) (Fig. 2e).SBS-RT represented 28.6% of the mutations acquired in RT (mean 679, range 499-1,167) and it was occasionally associated with coding mutations in driver genes (EP300 and CIITA) (Fig. 2f, Extended Data Fig. 5g and Supplementary Table 16).By applying a high-coverage, unique molecular identifier (UMI)-based next-generation sequencing (NGS) approach in longitudinal samples of patients 12, 19 and 63 (Supplementary Table 18), we observed that mutations of the RT subclones found in the main peaks of the SBS-RT were mainly identified in samples collected after bendamustine or chlorambucil therapy, whereas Chromothripsis (5, 9, 14, 15) Chromothripsis (4, 8, 12) Chromothripsis (1) Other complex (11) Non-complex Case 1,669 (RT) Time point: ",
"section_name": "New mutational processes in RT.",
"section_num": null
},
{
"section_content": "mutations not associated with SBS-RT were detected earlier during the disease course (Fig. 2g and Extended Data Fig. 5h).These results suggest a causal link between the exposure to these drugs and SBS-RT.The finding of SBS-melphalan, SBS-ganciclovir and SBS-RT in RT argues in favor of a single-cell expansion model for RT; a single cell that can carry the footprints of cancer therapies (Fig. 2h).Contrarily, the lack of SBS-RT in the 27 post-treatment CLL samples (7 patients treated with bendamustine or chlorambucil) suggests that CLL relapse might be driven by the simultaneous expansion of different subclones, hindering the detection of SBS-RT through bulk sequencing 34,36 . RT subclones also acquired kataegis, mainly within the immunoglobulin loci, attributed to activation-induced cytidine deaminase (AID) activity (SBS84 and SBS85) 29,32 (Fig. 2i and Extended Data Fig. 4).These kataegis led to the acquisition of mutations in the rearranged V(D)J gene in five RT cases (one after CIT and four targeted therapies) (Fig. 2i, Extended Data Fig. 5i,j and Supplementary Table 19).This canonical AID activity in RT is concordant with the acquisition of SBS9 mutations in two RT samples (4,686 (CIT) and 3,495 (targeted therapies)) and SVs mediated by aberrant class-switch recombination or somatic hypermutation in six RT (one before therapy, two CIT and three new agents), which targeted MYC, MYCN, TRAF3 and CCND3 (Fig. 1c and Supplementary Table 2). SBS-RT mutations were found in CLL samples before the transformation in patient 3,299 although it was only present in the RT subclone (Fig. 2c,e).SBS-RT was also found in two different subclones in case 12 and 19.We speculated that these secondary subclones with SBS-RT (named 'RT-like' subclones) could correspond to the single-cell expansion of a 'transformed' cell that could have been missed by the routine analysis (Fig. 2e).The reanalysis of flow cytometry data available for case 12 detected two cell populations at time point (T) 4 differing in size and surface markers (likely CLL and RT-like subclones), whereas at T5 we detected an additional population of large cells (RT subclone, 0.2% cells) that expanded at T6, substituting the previous large cell population (RT-like subclone) (Fig. 2j and Extended Data Fig. 5k-m).WGS analysis showed that the RT-like and RT subclones diverged from a cell carrying a deletion of CDKN2A/B and truncation of CREBBP, each acquiring more than 2,100 specific mutations (Fig. 2e,j). Altogether, these findings show that RT may arise simultaneously from different subclones and that such subclones can be detectable time before their final expansion and clinical manifestation.The identification of mutations in RT associated with early-in-time CLL therapies demonstrates that RT emerges from the clonal expansion of a single cell previously exposed to these therapies. ",
"section_name": "NATuRE MEDiCiNE",
"section_num": null
},
{
"section_content": "The WGS-based subclonal phylogeny of the nine patients with fully characterized longitudinal samples predicted that the RT subclone was present at low cancer cell fraction (CCF) in the preceding CLL samples in five (56%) patients and only detected at time of transformation in the remaining four (44%) (Fig. 3a).Indeed, the RT subclone was detected at time of CLL diagnosis in three of five patients, remained stable at a minute size (<1%) for 6-19 years of natural and treatment-influenced CLL course and expanded at the moment of clinical manifestations (patients 12, 19 and 63) (Fig. 3a).In the other two patients, the RT subclone was also detected in the first CLL sample analyzed but rapidly expanded driving the RT 0.6 and 3.5 years later in patients 3,034 and 3,299 (RT-PLL), respectively (Fig. 3a and Extended Data Fig. 6). We next performed single-cell DNA sequencing (scDNA-seq) of 32 genes in 16 longitudinal samples of 4 patients (12, 19, 365 and 3,299) to validate these evolutionary histories of RT (202,210 cells passing filters, mean of 12,638 cells per sample; Fig. 1a, Supplementary Fig. 2 and Supplementary Table 20).Focusing on patient 19 with a time lapse of 14.4 years from diagnosis to RT (Fig. 3b), the RT subclone (subclone 5) at transformation (T6) carried CDKN2A/B and TP53 (p.G245D) alterations, whereas the main CLL subclones driving the relapse after therapy at T4 and T5 harbored a different TP53 mutation (p.I195T; subclones 3 and 4).The WGS predicted the presence of all these subclones at CLL diagnosis (T1).Using scDNA-seq we identified two small populations accounting for 0.1% of cells carrying the TP53 p.I195T and p.G245D mutations, respectively, at T1, which were also detected at relapse 7.2 years later (T3).The subclone carrying TP53 p.I195T expanded to dominate the second relapse after 3.7 years at T4 and T5 but was substituted by the subclone carrying TP53 p.G245D at T6 in the RT 14.4 years after diagnosis.All these subclones carried the SF3B1 and NOTCH1 mutations of the initial CLL subclone (Fig. 3c and Supplementary Table 20).The scDNA-seq of the three additional cases also corroborated the phylogenies and most of the dynamics inferred from WGS (Extended Data Fig. 6a).These results suggest that CLL evolution to RT is characterized by an early driver diversification probably generated before diagnosis, consistent with the early immunogenetic and DNA methylation diversification previously reported in CLL [37][38][39] and that RT may emerge by a selection of pre-existing subclones carrying potent driver mutations rather than a de novo acquisition of leading clones. As we identified five cases of RT carrying specific mutations in the immunoglobulin genes by WGS (Fig. 2i), we analyzed whether these immunoglobulin-based RT subclones were already present at CLL diagnosis using high-coverage NGS in patients 12 and 3,495 (Supplementary Table 21).Focusing on patient 3,495, for which the lack of germline material precluded our phylogenetic analyses, the RT occurring after treatment with ibrutinib harbored two new V(D)J mutations generating an unproductive IGH gene.NGS identified 0.002% sequences carrying the same two mutations at CLL diagnosis 1.72 years before (Fig. 3d).We also observed the expansion of additional unproductive subclones accounting for 11.8% of all sequences at time of RT, suggesting that BCR-independent subclones may have a proliferative advantage under therapy with BCR inhibitors (Fig. 3d).Similar results were found in patient 12 in which the V(D)J sequence of RT carrying a new mutation was already identified at CLL diagnosis 19.5 years before at DNA and RNA level (Fig. 3e).As the immunogenetic features represent a faithful imprint of the B cell of origin, the early identification of the same immunogenetic subclone provides further evidence for an early seeding of RT. We finally tracked RT subclones during the disease course using single-cell RNA sequencing (scRNA-seq) of 19 longitudinal samples of five patients (24,800 tumor cells passing filters, mean of 1,305 cells per sample; Fig. 1a and Supplementary Table 22).As expected, RT and CLL cells had remarkably different gene expression profiles (Fig. 3f and Extended Data Fig. 7a-d).The transcriptome of CLL cells was dominated by three main clusters identified across patients and characterized by different expression of CXCR4, CD27 and MIR155HG, respectively, which may represent the recirculation of CLL cells between peripheral blood and lymph nodes [40][41][42] (Fig. 3f,g and Extended Data Fig. 7a-d).Contrarily, RT intraclonal heterogeneity was mainly related to distinct proliferative capacities with a cluster of cells showing high MKI67 and PCNA expression as well as high S and G2M cell-cycle phase scores.The remaining RT clusters were characterized by the expression of different marker genes among patients, including CCND2, MIR155HG and TP53INP1 (Fig. 3f-h and Extended Data Fig. 7a-d).When considering each time point separately, we detected RT cells in all CLL samples before transformation in patient 12, 19, 63 and 3,299 but not in patient 365 (Fig. 3i and Extended Data Fig. 7a-i).The presence and dynamics of these RT subclones according to their transcriptomic profile recapitulated the findings obtained by WGS, scDNA-seq and immunoglobulin analyses in all five patients, suggesting that they captured the same cells.Indeed, using scRNA-seq we could identify the CNAs involved in simple and complex structural alterations found at time of RT by WGS already in the dormant RT cells at CLL diagnosis and subsequent time points before their final expansion (Fig. 3j and Extended Data Fig. 8).These findings suggest an early acquisition of SVs, including chromothripsis and transcriptomic identity in RT. To validate our observations, we reanalyzed the longitudinal scRNA-seq dataset from Penter et al. 43 consisting of nine patients with CLL, one of which developed RT.In this case, we identified RT cells in the CLL sample collected 1.6 years before the RT (Extended Data Fig. 7j).Overall, our integrative analyses uncovered a widespread early seeding of RT cells up to 19 years before their expansion and clinical manifestation. OXPHOS high -BCR low transcriptional axis of RT.To understand the transcriptomic evolution from CLL to RT and its epigenomic regulation, we integrated genome-wide profiles of DNA methylation, chromatin activation (H3K27ac) and chromatin accessibility (ATAC-seq) with bulk RNA-seq and scRNA-seq of multiple longitudinal samples of six patients treated with BCR inhibitors (Fig. 1a).The DNA methylome of RT mainly reflected the naive and memory-like B cell derivation of their CLL counterpart, whereas chromatin activation and accessibility were remarkably different upon transformation (Fig. 4a).We identified 150 regions with increased H3K27ac and 426 regions that gained accessibility in RT (Fig. 4b, Extended Data Fig. 9a and Supplementary Tables 7 and8).These de novo active regions were enriched in transcription factor (TF) families different from those known to modulate the epigenome of CLL 44 .Among them, 24 were enriched and upregulated in RT (Supplementary Table 7).The top TF was TEAD4, which 2 3 1 2 3 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 1 2 1 2 1 2 3 4 activates genes involved in oxidative phosphorylation (OXPHOS) through the mTOR pathway 45 and co-operates with MYCN 46 .Additional TFs were related to MYC (MAZ), proliferation/cell cycle (E2F family) or IRF family, among others (Fig. 4c).Notably, high IRF4 levels seem to attenuate BCR signaling in CLL 47 , whereas they are necessary to induce MYC target genes, OXPHOS and glycolysis in activated healthy B cells 48 . The RNA-seq analysis, excluding cases 19 and 3,299 (RT-PLL) due to their intermediate transcriptomic profile, identified 2,248 differentially expressed genes (DEGs) between RT and CLL (1,439 upregulated and 809 downregulated) (Fig. 4a,d,e, Extended Data Fig. 10a and Supplementary Tables 11 and23).A remarkable fraction of upregulated/downregulated genes overlapped with regions with the respective increase/decrease of H3K27ac (20%) and chromatin accessibility (16%) at RT (Fig. 4d and Extended Data Fig. 9b).Contrarily, only 4% of the DEGs overlapped with any of the 2,341 differentially methylated CpGs (DMCs) between RT and CLL, emphasizing the limited effect of DNA methylation on gene regulation 49 .Most DMCs were hypomethylated at RT (2,112 of 2,341; 90%), found in open sea and intergenic regions and correlated with the proliferative history of the cells measured by the epiCMIT score 49 (1,681; 72%), which increased during CLL evolution and at RT (Fig. 4d,f, Extended Data Fig. 9c-g and Supplementary Table 6). Genes upregulated in RT involved pathways that seem independent of BCR signaling such as Wnt (WNT5A and others) 50 , Toll-like receptors (TLR9 among others) 51 and a number of cyclin-dependent kinases.Downregulated genes included, among others, CXCR4, HLA-A/B and chromatin remodelers also targeted by genetic alterations in some cases (Fig. 4d and Extended Data Fig. 10b,c).Gene sets modulated by gene expression in RT were in harmony with the identified chromatin-based changes and included upregulation of E2F targets, G2M checkpoints, MYC targets, MTORC1 signaling, OXPHOS, mitochondrial translation, glycolysis, reactive oxygen species and DNA repair pathways, among others.In addition, RT showed downmodulation of BCR signaling (Fig. 4g,h, Extended Data Fig. 10d and Supplementary Table 11).The OXPHOS high -BCR low pattern observed by bulk RNA-seq in RT was further refined using scRNA-seq: two of five tumors had OXPHOS high -BCR low (12 and 63, although the latter showed some intercluster variability), the two M-CLL carrying IGLV3-21 R110 had RT with BCR expression similar to CLL and were OXPHOS high -BCR normal (365) or OXPHOS normal -BCR normal (19) and the RT-PLL (3,299) was OXPHOS low -BCR low (Fig. 4i, Extended Data Fig. 10e-j and Supplementary Table 23).In addition, the scRNA-seq analysis showed that the OXPHOS/ BCR profiles of RT were already identified in the early dormant RT cells, suggesting that they might represent an intrinsic characteristic of RT cells rather than being modulated by BCR inhibitors (Fig. 4j and Extended Data Fig. 10g-j).To expand these observations, we measured the expression of OXPHOS and BCR pathways in the scRNA-seq dataset from Penter et al. 43 developed RT in the absence of any therapy, showed a remarkably higher OXPHOS and slightly lower BCR expression at time of RT compared to CLL (Fig. 4k and Extended Data Fig. 10k,l).Overall, the epigenome and transcriptome of RT converge to an OXPHOS high -BCR low axis reminiscent of that observed in the de novo DLBCL subtype characterized by high OXPHOS (DLBCL-OXPHOS) and insensitive to BCR inhibition [52][53][54] .This axis might explain the selection and rapid expansion of small RT subclones under therapy with BCR inhibitors. OXPHOS and BCR activity in RT.We next validated experimentally the OXPHOS and BCR activity of RT in samples of patients 12, 19 and 63.Respirometry assays confirmed that OXPHOS high RT cells (patients 12 and 63) had a 3.5-fold higher oxygen consumption at routine respiration and fivefold higher electron transfer system capacity (ETC) compared to CLL.In addition, OXPHOS normal RT (patient 19) showed a routine oxygen consumption similar to CLL, although also had a relatively higher ETC than its CLL counterpart (Fig. 5a, Supplementary Fig. 3a-d and Supplementary Table 24).BCR signaling measured by Ca 2+ mobilization upon BCR stimulation with IgM showed that BCR low RT cells (patients 12 and 63) had a lower Ca 2+ flux compared to CLL, which contrasted with the higher flux observed in the BCR normal RT cells of patient 19, concordant with its IGLV3-21 R110 mutation 27 (Fig. 5b, Supplementary Fig. 4a,b and Supplementary Table 25). To determine the biological effect of OXPHOS high in RT, we performed in vitro proliferation assays using IACS-010759 (100 nM), an OXPHOS inhibitor that targets mitochondrial complex I (Supplementary Figs.3e and4c and Supplementary Table 25).OXPHOS high RT (patients 12 and 63) had a higher proliferation at 72 h compared to OXPHOS normal RT (patients 19) and all of them were higher than their respective CLL.OXPHOS inhibition resulted in a marked decrease in proliferation in OXPHOS high RT (mean 49.1%), which contrasted with that observed in OXPHOS normal RT (2.2% decrease) and CLL (23.2% decrease) (Fig. 5c and Supplementary Fig. 4d).Overall, these results confirm the role of OXPHOS high phenotype in high proliferation of RT and suggest its potential therapeutic value in RT as proposed for other neoplasms [53][54][55][56][57] . ",
"section_name": "Dormant seeds of RT at CLL diagnosis.",
"section_num": null
},
{
"section_content": "The genome of RT is characterized by a compendium of driver alterations in cell cycle, MYC, NOTCH and NF-κB pathways, frequently targeted in single catastrophic events and by the footprints of early-in-time, treatment-related, mutational processes, including the new SBS-RT potentially associated with bendamustine and chlorambucil exposure.A very early diversification of CLL leads to emergence of RT cells with fully assembled genomic, immunogenetic and transcriptomic profiles already at CLL diagnosis up to 19 years before the clonal explosion associated with the clinical transformation.RT cells have a notable shift in chromatin configuration and transcriptional program that converges into activation of the OXPHOS pathway and downregulation of BCR signaling, the latter potentially compensated by activating Toll-like, MYC and MAPK pathways 17,51,58,59 .The rapid expansion of RT subclones under treatment with BCR inhibitors is consistent with its low BCR signaling, except when carrying the IGLV3-21 R110 and further supported by the increased number of subclones carrying unproductive immunoglobulin genes and the development of RT with plasmablastic differentiation, a cell type independent of BCR signaling 60 .Finally, we also uncovered that OXPHOS inhibition reduced the proliferation of RT cells in vitro, a finding worth exploring in future therapeutic strategies 55,57 . In conclusion, our comprehensive characterization of CLL evolution toward RT has revealed new genomic drivers and epigenomic reconfiguration with very early emergence of subclones driving late stages of cancer evolution, which may set the basis for developing single-cell-based predictive strategies.Furthermore, this study also identifies new RT-specific therapeutic targets and suggests that early intervention to eradicate dormant RT subclones may prevent the future development of this lethal complication of CLL. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "",
"section_name": "NATuRE MEDiCiNE",
"section_num": null
},
{
"section_content": "Consent and sample processing.Written informed consent was obtained from all patients.The study was approved by the Hospital Clinic of Barcelona Ethics Committee.Tumor DNA was extracted from tumor cells purified from fresh/cryopreserved mononuclear cells, frozen lymph nodes or formalin-fixed paraffin-embedded (FFPE) tissue (n = 1, CLL sample of patient 1,669).Germline DNA was obtained from the non-tumoral purified cell fraction in 12 cases.In two patients (1,523 and 4,675) who had received allogeneic stem-cell transplant before RT, germline DNA of the donor was also collected.All extractions were performed using appropriate QIAGEN kits (QIAamp DNA Blood Maxi kit, cat.no.51194; QIAamp DNA Mini kit, cat.no.51304; and AllPrep DNA/RNA FFPE kit, cat.no.80234).Tumor RNA was obtained from tumor cells purified from fresh/cryopreserved mononuclear cells with TRIzol reagent (Invitrogen, cat.no.15596026). A specific flow cytometry analysis was conducted on peripheral blood samples of patient 12, which were stained with the Lymphocyte Screening Tube according to EuroFlow protocols (https://www.euroflow.org/protocols).At least 100,000 cells were acquired in a FACSCanto II instrument.Analysis was conducted using the Infinicyt 2.0 software.The sequential gating analysis was as follows: singlet identification in a FSC-W versus FSC-H plot; leukocyte identification in SSC-A versus CD45 (V500-C) plot and FSC-A versus SSC-A; lymphocytes identified as SSC-A low and CD45 high and back-gated in FSC-A versus SSC-A to exclude monocytes; in the lymphocyte gate, T cells were identified as CD3 + cells in SSC-A versus CD3 (APC) followed by sequentially distinguishing TCRγδ + T cells, CD4 T cells and CD8 T cells; after excluding T cells, B cells were selected in a SSC-A versus CD19 (PE-Cy7), followed by inspection of CD19 (PECy7) versus CD20 (PacB), CD5 (PerCPCy5.5)versus CD20 (PacB) and CD20 (PacB) versus CD38 (APC-H7) plots to evaluate the expression of these B cell markers and the assignation of κ and λ expression in a plot of IgK (PE) versus IgL (FITC); after excluding B cells, natural killer cells were identified in a SSC-A versus CD56 (PE) plot followed by SSC-A versus CD38 (APC-H7) plot. ",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Library preparation and sequencing.All samples available were subjected to WGS except the FFPE CLL, which was analyzed by whole-exome sequencing (WES).WGS libraries were performed using the Kapa Library Preparation kit (Roche, cat.no.07961901001), TruSeq DNA PCR-Free kit (Illumina, cat.no.20015963) or TruSeq DNA Nano protocol (Illumina, cat.no.20015965) and sequenced on a HiSeq 2000/4000/X Ten (2 × 126 bp or 2 × 151 bp) or NovaSeq 6000 (2 × 151 bp) instrument (Illumina).WES was performed using the SureSelect Human All Exon V5 (Agilent Technologies, cat. no.5190-6209 and G9611B) coupled with a KAPA Hyper Prep kit (Roche, cat.no.07962363001) for the DNA pre-capture library.Sequencing was performed on a HiSeq 2000 (2 × 101 bp).We also included WGS of three published CLL/germline pairs (patients 12, 19 and 63) 28 (Supplementary Table 1). General considerations.Overall, 12 patients had a complete dataset (germline, CLL and RT samples), 6 patients lacked germline DNA and 1 patient had only the RT sample (case 4,676).We conducted tumor versus normal analyses in cases with a complete dataset.For the six patients lacking the germline sample, we used the CLL samples as 'normal' to identify SNV acquired at RT for mutational signature analyses.In addition, tumor-only analyses were conducted in these CLL and RT samples, as well as in the patient with only a RT sample available, to identify driver gene mutations and genome-wide CNAs (Supplementary Table 1). Read mapping and quality control.Reads were mapped to the human reference genome (GRCh37) using the BWA-MEM algorithm (v.0.7.15) 61 .BAM files were generated and optical/PCR duplicates flagged using biobambam2 (v.2.0.65, https:// gitlab.com/german.tischler/biobambam2).FastQC (v.0.11.5, www.bioinformatics.babraham.ac.uk/projects/fastqc) and Picard (v.2.10.2, https://broadinstitute.github.io/picard) were used to extract quality control metrics.Mean coverage was 33× and 119× for WGS and WES, respectively (Supplementary Table 1). Immunoglobulin gene characterization.Immunoglobulin gene rearrangements were characterized using IgCaller (v.1.2) 62 .The rearranged sequences obtained were reviewed on the Integrative Genomics Viewer (IGV; v.2.9.2) 63 and annotated using IMGT/V-QUEST (https://www.imgt.org/IMGT_vquest)and ARResT/ AssignSubsets (http://bat.infspire.org/arrest/assignsubsets). Tumor versus normal SNVs and indel calling.SNVs were called using Sidrón 28 , CaVEMan (cgpCaVEManWrapper, v.1.12.0) 64 , Mutect2 (Genome Analysis Toolkit (GATK) v.4.0.2.0) 65 and MuSE (v.1.0rc) 66 and normalized using bcftools (v.1.8) 67.Variants detected by CaVEMan with more than half of the mutant reads clipped (CLPM > 0) and with supporting reads with a median alignment score (ASMD) <90, <120 or <140 for sequencing read lengths of 100, 125 or 150 bp, respectively, were excluded.Variants called by Mutect2 with MMQ < 60 were eliminated.Mutations detected by at least two algorithms were considered.Short insertions/ deletions (indels) were called by SMuFin (v.0.9.4) 68 , Pindel (cgpPindel, v.2.2.3) 69 , SvABA (v.7.0.2) 70 , Mutect2 (GATK v.4.0.2.0) 65 and Platypus (v.0.8.1) 71 .The somaticMutationDetector.py script (https://github.com/andyrimmer/Platypus/blob/master/extensions/Cancer/somaticMutationDetector.py) was used to identify somatic indels called by Platypus.Indels were left-aligned and normalized using bcftools 67 .Indels with MMQ < 60, MQ < 60 and MAPQ < 60 for Mutect2, Platypus and SvABA, respectively, were removed.Only indels identified by at least two algorithms were retained.Annotation of mutations was performed using snpEff/ snpSift (v.4.3t) 72 and GRCh37.p13.RefSeq as a reference.This approach showed a 93% specificity and 88% sensitivity when benchmarked against the mutations found at a VAF >10% in our previous high-coverage NGS study 73 . Tumor-only SNVs and indel calling.Tumor-only variant calling was restricted to coding regions of 243 genes described as drivers in CLL and other B cell lymphomas (Supplementary Table 10).Mini-BAM files were obtained using Picard tools and variant calling was performed using Mutect2 (GATK v.4.0.4.0) 65 , VarScan2 (v.2.4.3) 74 , VarDictJava (v.1.4) 75, LoFreq (v.2.1.3.1) 76, outLyzer (v.1.0) 77nd freebayes (v.1.1.0,https://github.com/freebayes/freebayes). Variants were normalized using bcftools (v.1.9) 67and annotated using snpEff/snpSift (v.4.3t) 72 .Only non-synonymous variants that were identified as PASS by ≥2 algorithms were considered.Variants reported in 1000 Genomes Project, ExAC or gnomAD with a population frequency >1% or reported as germline in our ICGC database of 506 WES/WGS 28 were considered as polymorphisms. Tumor versus normal CNA calling.CNAs were called using Battenberg (cgpBattenberg, v.3.2.2) 78 and ASCAT (ascatNgs, v.4.1.0) 79.CNAs within any of the immunoglobulin loci were not considered.We used the tumor purities obtained by Battenberg in downstream analyses.The median tumor cell content was 91.5% (Supplementary Table 1). Tumor-only CNA calling.CNAs were extracted using CNVkit (v.0.9.3) 80 .CNAs <500 kb, with an absolute log 2 copy ratio (log 2 CR) < 0.3 or located within any of the immunoglobulin loci were removed.CNAs were classified as gains if log 2 CR > 0.3, deletions if log 2 CR < -0.3, high-copy gains if log 2 CR > 1.1 and homozygous deletions if log 2 CR < -1.1.The log 2 CR cutoff was set to 0.15 for two samples with low tumor cell content (102-01-01TD and 4690-03-01BD).To avoid a high segmentation of the CNA profile, CNAs belonging to the same class were merged if they were separated by <1 Mb and had an absolute log 2 CR difference <0.25. Array-based CNA calling in FFPE.CNAs were examined in the FFPE CLL sample using the Oncoscan CNV FFPE Assay kit (Thermo Fisher Scientific, cat.no.902695) and analyzed using Nexus 9.0 software (Biodiscovery). Tumor versus normal SV calling.SVs were extracted using SMuFin (v.0.9.4) 68 , BRASS (v.6.0.5) 81 , SvABA (v.7.0.2) 70 and DELLY2 (v.0.8.1) 82 .SVs identified were intersected considering a window of 300 bp around break points.We kept for downstream analyses the SVs identified by at least two programs if at least one of the algorithms called the alteration with high quality (MAPQ ≥ 90 for BRASS, MAPQ = 60 for SvABA and DELLY2).In addition, IgCaller (v.1.2) 62 was used to call SVs within any of the immunoglobulin loci.All SVs were visually inspected using IGV 63 .SVs were categorized into simple or complex events.Chromothripsis 83 was defined as ≥7 oscillating changes between two or three copy number states or the presence of >7 SV break points occurring in a single chromosome and supported by additional criteria 83,84 .Chromoplexy was determined by the presence of ≥3 chained chromosomal rearrangements, where chains were identified using a window of 50 kb 85,86 .Cycles of templated insertions were defined as copy number gains in ≥3 chromosomes linked by SVs 87 .Breakage-fusion bridge cycles were defined as patterns of focal copy number increases and fold-back inversions, together with telomeric deletions.Chains of rearrangements having >2 SVs and not fulfilling any of the previous criteria were classified as 'other complex events' .Chromothripsis and 'other complex events' were subcategorized according to the number of chromosomes involved.The longitudinal nature of our dataset allowed us to refine the obtained classification based on the presence of the involved alterations in each time point analyzed. Patients who underwent allogenic stem-cell transplant.In these patients, we conducted tumor versus patient's germline and tumor versus donor's germline variant calling in parallel.Only the intersection of variants identified was considered. ",
"section_name": "WGS and WES.",
"section_num": null
},
{
"section_content": "were automatically added to the samples of additional time point(s) if at least one high-quality read with the mutation was found in the BAM file (alleleCounter v.4.0.0, parameters: min_map_qual = 35; and min_base_qual = 20).Similarly, indels and SVs detected in one sample were added in the additional time point(s) if any of the algorithms detected the alteration, regardless of its filters. ",
"section_name": "Rescue of alterations based on longitudinal information. SNVs called in one sample",
"section_num": null
},
{
"section_content": "A Markov chain Monte Carlo sampler for a Dirichlet process mixture model was used to infer putative subclones, to assign mutations to subclones and to estimate the subclone frequencies in each sample from the SNV read counts, copy number states and tumor purities (Supplementary Table 17) 78,88 .Clusters with <100 mutations were excluded.The phylogenetic relationships between subclones were identified following the NATuRE MEDICINE | www.nature.com/naturemedicine'pigeonhole principle' , which was relaxed using a case-specific 'tolerated error' 88 . Clusters not assigned to the reconstructed phylogenetic tree were excluded.Fish plots were generated using the TimeScape R package (v.1.6.0).The CCF of indels was calculated integrating read counts, CNAs and tumor purity 89 .Driver indels subjected to validation by scDNA-seq and/or relevant to the tumor phylogeny were manually assigned to subclones.Similarly, driver CNAs relevant to the phylogeny were manually assigned.Seven SNVs found in TP53/ATM overlapping with CNAs were manually assigned to the most likely subclone as they were not automatically assigned by the Dirichlet process and were subjected to scDNA-seq (Supplementary Table 9).Mutational signatures.We studied mutational signatures acting genome-wide and in localized regions (inter-mutation distance ≤1Kb) 29,32 .We integrated the mutations identified in this CLL/RT cohort together with those of 147 CLL treatment-naive samples (ICGC-CLL) 28 and 27 new CLL collected at relapse post-treatment (mean coverage 31.5×;Supplementary Table 15).The WGS of these two additional cohorts was (re-)analyzed using our current bioinformatic pipeline (Supplementary Table 12).Mutational signatures were analyzed for SNVs or single-base substitutions (SBSs) according to their 5′ and 3′ flanking bases following three steps 30 : 1. Extraction: de novo signature extraction was performed using a hierarchical Dirichlet process (HDP, v.0.1.5;https://github.com/nicolaroberts/hdp),SignatureAnalyzer (v.0.0.7) 90 , SigProfiler (SigProfilerExtractor, v.1.0.8) 32 and sigfit (v.2.0.0; https://github.com/kgori/sigfit).HDP was run with four independent posterior sampling chains, followed by 20,000 burn-in iterations and the collection of 200 posterior samples off each chain with 200 iterations between each.SigProfiler was run with 1,000 iterations and a maximum of ten extracted signatures.Similarly, sigfit was run to extract five signatures with 10,000 burn-in iterations and 20,000 sampling iterations.2. Assignment: each extracted signature was assigned to a given COSMIC signature (v.3.2) 32 if their cosine similarity was >0.85.Otherwise, the extracted signature was decomposed into 'n' COSMIC signatures using an expectation maximization (EM) algorithm 91 .The EM algorithm was first run using the COSMIC signatures identified in the previous step.If their cosine similarity was <0.85, we ran the EM algorithm, including all signatures reported in COSMIC and by Kucab et al. 33 (55 mutational signatures related to environmental agents).Three exceptions were made: (1) we combined two HDP signatures that together constituted COSMIC signature SBS5 to avoid splitting of signatures (Extended Data Fig. 4a); (2) APOBEC signatures (SBS2 and SBS13) were favored to be assigned to one of the signatures extracted by HDP and SignatureAnalyzer although it was not the best EM solution probably because they were only found in one sample, which impaired a clean extraction of the signatures (Extended Data Fig. 4f); and (3) one signature extracted by HDP and SignatureAnalyzer was directly assigned to the mutational signature associated with ganciclovir treatment 35 (cosine similarity 0.987 and 0.993, respectively) (Extended Data Fig. 4).The new SBS-RT extracted by HDP was considered for downstream analyses as it had less background noise than the one extracted by SignatureAnalyzer, favoring a higher specificity during the fitting step.Similarly, the SBS-ganciclovir extracted by HDP was used in downstream analyses (Extended Data Fig. 4).We also performed a detailed review to remove signatures susceptible of being originated due to sequencing artifacts (Supplementary Table 13).3. Fitting: we used a fitting approach (MutationalPatterns, v.3.0.1) to measure the contribution of each mutational signature in each sample.Based on (1) the de novo identification of the therapy-related SBS-ganciclovir and ( 2) that two patients received melphalan before RT, the mutational signature associated with melphalan therapy 34 was also included in this step.To avoid the so-called inter-sample bleeding effect 30 , we iteratively removed the less-contributing signature if its removal decreased the cosine similarity between the original and reconstructed 96-profile <0.01 (ref. 32).SBS1 and SBS5 were added if addition improved the cosine similarity 32 .Similarly, SBS9 was added in CLL/RT samples classified as M-CLL if addition improved the cosine similarity.We also ran mSigAct (v.2.1.1;https://github.com/steverozen/mSigAct)to confirm the presence/absence of SBS-melphalan (Supplementary Table 15).To assess the contribution of each signature to each subclone we followed the same fitting strategy but (1) considered only the signatures that were present in the corresponding sample and (2) removed the final step of adding SBS9 in M-CLL to avoid its addition in multiple subclones with low evidence. Genomic locations and strand bias.We assessed the contribution of SBS-RT to coding SNVs in RT subclones (also including cases in which the CLL sample was used as a 'germline') by calculating the probability that a given mutation was caused by SBS-RT.To perform this calculation, we considered the signatures present in the subclone/sample and their signature profile 92 .The reference epigenomes of CLL 44 were used to explore the contribution of the mutational processes in different regulatory regions.We simplified the described chromatin states in four categories: heterochromatin (H3K9me3_Repressed, Heterochromatin Low_Signal), polycomb (Posied_Promoter, H3K27me3_Repressed), enhancer/promoter (Active_Promoter, Strong_Enhancer1, Weak_Promoter, Weak_Enhancer, Strong_Enhancer) and transcription (Transcription_Transition, Weak_Transcription, Transcription_ Elongation).We also mapped the activity of mutational processes in early/late replication regions of the genome considering peaks/valleys of early/late replication as those regions of ≥1 kb with absolute replication timing >0.5 (ref. 93).All SNVs of the CLL and RT subclones were classified in any of the four chromatin states and early/late replication regions before fitting mutational signatures.A cutoff of 0.005 was used to remove the less-contributing signature during the fitting step.We also generated replication and transcriptional strand bias profiles of the RT-specific mutations using the MutationalPatterns R package 34 .The replication strand was annotated based on the left/right replication direction of the timing transition regions 94 .The transcriptional strand was annotated using the TxDb.Hsapiens.UCSC.hg19.knownGeneR package (v.3.2.2).Finally, kataegis was defined as a genomic region having six or more mutations with an average inter-mutation distance ≤1 kb.-read structure = 16M+T 16M+T,single-tag = RX, -molecular-index-tags = ZA ZB).Template read was converted to FASTQ with Picard's SamToFastq.Template reads were mapped against the human reference genome (GRCh37) and reads were merged with the UMI information using Picard's MergeBamAlignment.Finally, reads were grouped by UMI and a consensus was called using fgbio GroupReadsByUmi (parameters were -strategy = adjacency, -edits = 1, -min-map = 10) and CallMolecularConsensusReads (parameters were -min-reads = 3), respectively.A minimum of three reads was required to create a UMI-based final read.Final reads were converted back to FASTQ using Picard's SamToFastq and mapped against the reference genome using BWA-MEM (v.0.7.15) 61 .Mean coverage was determined using Picard's CollectTargetedPcrMetrics (parameters: CLIP_OVERLAPPING_ READS = true, MINIMUM_MAPPING_QUALITY = 15 MINIMUM_BASE_ QUALITY = 15).Read counts were collected at all targeted genomic positions for all samples using bcftools mpileup (v.1.8,parameters: -B -Q 13 -q 10 -d 100,000 -a FORMAT/DP,FORMAT/AD,FORMAT/ADF,FORMAT/ADR -O v) 67 .Allele positions lacking mutations by WGS were used to model the background sequencing noise, which was unified according to the trinucleotide context of each possible mutation.Mutations of interest were annotated as high confidence when their frequency was above the background noise with a probability of 95%.DNA methylation.Data generation and processing.DNA methylation data of 39 samples was generated using EPIC BeadChips (Illumina).These samples included different healthy B cell subpopulations (naive B cells (NBCs), n = 2; germinal center B cells (GCs), n = 1; memory B cells (MBCs), n = 3; tonsillar plasma cells (tPCs), n = 1); CLL samples without evidence of RT (n = 12) and longitudinal CLL/ RT samples (n = 20) (Supplementary Table 6).R and core Bioconductor packages, including minfi (v.1.34.0) 96 , were used to integrate and normalize DNA methylation data 49 .We removed non-CpG probes, CpGs representing single nucleotide polymorphisms, CpGs with individual-specific methylation previously reported in B cells, CpGs in sex chromosomes and CpGs with a detection P value >0.01 in >10% of the samples.The data were normalized using the SWAN algorithm and CpGs were annotated using the IlluminaHumanMethylationEPICanno.ilm10b4.hg19 package (v.0.6).Tumor cell content of each sample was inferred from DNA methylation 49 and samples with a tumor cell content <60% were excluded.After all filtering criteria, we retained 33 samples (NBCs, n = 2; GCs, n = 1; MBCs, n = 3; tPCs, n = 1; CLL controls, n = 12; CLL/RT samples, n = 14 (six patients); Supplementary Table 6). ",
"section_name": "WGS-based subclonal reconstruction.",
"section_num": null
},
{
"section_content": "",
"section_name": "High",
"section_num": null
},
{
"section_content": "Differential analyses, CLL epitypes and epiCMIT.We compared the DNA methylation status of each CpG to the mean of such CpGs in NBCs to calculate the number of hyper-and hypomethylation changes per CLL/RT sample.Changes in each sample were defined based on a minimum difference of 0.25 methylation. To perform a differential analysis between CLL and RT, we compared the DNA methylation of each CpG in each CLL sample (first available time point used) versus their respective RT sample.Differentially methylated CpGs were considered as those showing a minimum difference of 0.25 in at least four of the five longitudinal cases of RT versus CLL analyzed (Supplementary Table 6).The epigenetic subtypes (epitypes) and epiCMIT score for each CLL and RT sample were calculated 49 . ChIP-seq of H3K27ac and ATAC-seq.Data generation.ChIP-seq of H3K27ac and ATAC-seq data were generated as described in http://www.blueprint-epigenome. eu/index.cfm?p=7BF8A4B6-F4FE-861A-2AD57A08D63D0B58 (antibody anti H3K27ac, Diagenode, cat.no.C15410196/pAb-196-050, lot A1723-0041D; Supplementary Tables 7 and8).Libraries were sequenced on Illumina machines aiming at 60 million reads/sample (Supplementary Tables 7 and8). Read mapping and initial data processing.FASTQ files were aligned to the reference genome (GRCh38) using BWA-ALN (v.0.7.7, parameter: -q 5) 61 , duplicated reads were marked using Picard tools (v.2.8.1) and low-quality and duplicated reads were removed using SAMtools (v.1.3.1, parameters: -b -F 4 -q 5 -b -F 1,024) 67 .PhantomPeakQualTools (v.1.1.0)were used to generate wiggle plots and for extracting the predominant insert-size.Peaks were called using MACS2 (v.2.1.1.20160309,parameters for H3K27ac: -g hs -q 0.05 -keep-dup all -nomodel -extsize insert-size; parameters for ATAC-seq: -g hs -q 0.05-keep-dup all -f BAM -nomodel -shift -96 -extsize 200; no input control) 97 .Peaks with q values <1 × 10 -3 were included for downstream analyses.For each mark separately, a set of consensus peaks, including regions within chromosomes 1-22 and present in published healthy B cells 44 and CLL samples was generated by merging the locations of the separate peaks per individual sample.For ChIP-seq, the numbers of reads per sample per consensus peak were calculated using the genomecov function (bedtools, v.2.25.0).For ATAC-seq, the number of Tn5 transposase insertions per sample per consensus peak was calculated by first determining the estimated insertion sites (shifting the start of the first mate 4 bp downstream) before using the genomecov function.Variance stabilizing transformation (VST) values were calculated for all consensus peaks using DESeq2 (v.1.28.1) 98 , which were then corrected for the consensus SPOT score (the percentage of reads that fall within the consensus peaks) using the ComBat function (sva R package, v.3.36.0).To that purpose, the cell condition (tumor and different healthy B cell subtypes) was assigned to each sample and samples were clustered in 20 bins of 5% according to their consensus SPOT score.The bins on the extremes, which contained fewer than five samples, were joined with their neighboring bins to ensure that each bin contained five samples or more.PCA was generated using the corrected VST values of peaks that were present in more than one sample. ",
"section_name": "High",
"section_num": null
},
{
"section_content": "We first determined the regions with stable epigenetic profiles in the healthy B cell counterparts (NBCs and MBCs) by applying a threshold of s.d.< 0.8 with respect to the mean value.For all these NBC/MBC stable regions, we then calculated the log 2 FC between the mean of VST-corrected healthy B cell values and each of the tumor samples.Due to the data distribution variability, we applied slightly different thresholds of log 2 FC for each case (Supplementary Tables 7 and8).To identify regions changing in RT for each case individually, we selected the regions that presented substantial epigenetic changes as compared to the normal counterpart and to the previous CLL (absolute log 2 FC > 1).The ATAC-seq RT-specific signature encompassed differential regions common in two or more cases of RT, whereas the H3K27ac RT-specific signature included differential regions common in three or more cases.Potential protein-coding target genes were assigned to each of the RT-specific regions using two strategies.To identify close target genes, we took the overlap with the regions of genes of interest adding 2 kb upstream of their transcription start site.To identify distant target genes, we used Hi-C data from the GM12878 cell line and selected all genes located within the same topologically associated domain as the region of interest.We only considered DEGs identified by bulk RNA-seq (Supplementary Tables 7 and8). Transcription factor analysis.Enrichment for TF-binding sites was analyzed in chromatin accessible regions within the RT-specific active chromatin regions.Accessible peaks were determined as regions with presence of ATAC peaks in two or more RT cases.Enrichment analysis of known TF-binding motifs was performed using the AME tool (MEME suite) considering the non-redundant Homo sapiens 2020 Jaspar database and applying one-tailed Wilcoxon rank-sum tests with the maximum score of the sequence, a 0.01 FDR cutoff and a background formed by reference GRCh38 sequences extracted from the consensus ATAC-seq peaks (91,671 regions).We then established the occupancy of these motifs in RT and CLL by calculating the percentage of the target RT-specific active regions and of the regions with increased H3K27ac in CLL, respectively, which contained these motifs.Finally, we selected TFs presenting an occupancy difference between RT and CLL ≥ 10% and overexpressed in RT (bulk RNA-seq, log 2 FC > 0, adjusted P value <0.01). Bulk 11). Data analysis.Ribosomal RNA reads were filter out using SortMeRNA (v.4.3.2) 99 .Non-ribosomal reads were trimmed using Trimmomatic (v.0.38) 95 .Gene-level counts (GRCh38.p13,Ensembl release 100) were calculated using kallisto (v.0.46.1) 100 and tximport (v.1.14.2).A paired DEA was conducted using DESeq2 (v.1.26.0) 98 .Adjusted P value <0.01 and absolute log 2 (fold change) > 1 were used to identify DEGs.Gene set enrichment analysis (GSEA) was conducted using a pre-ranked gene list ordered by -log 10 (P) × (sign of fold change) using the 'GSEA' function (clusterProfiler R package, v.3.14.3).We focused on C2 (curated) and Hallmark gene sets from the Molecular Signatures Database (v.7.4) with a minimal size of 10 and maximal size of 250.Gene ontology (GO) GSEA was conducted using the pre-ranked gene list as input of the 'gseGO' function (clusterProfiler) focusing on biological processes.Redundancy in the output list of GO terms was removed using the 'simplify' function (cutoff of 0.35 101 .Genotypes were encoded as zero for wild-type, one for heterozygous mutation, two for homozygous mutation and three for missing data.∞SCITE was used to find the mutation tree that best fitted the genotypes observed and to assign cells into subclones.∞SCITE was run using a global sequencing error rate (false-positive rate) of 1% 102 , an estimated rate of non-mutated sites called as homozygous mutations of 0% and a patient-specific estimated rate of the allele dropout rate (false-negative rate).For each patient, the estimated rate of missed heterozygous mutations (dropout of the mutated allele) and the estimated rate of heterozygous mutations called as homozygous mutations (dropout of the normal allele) were calculated from germline single-nucleotide polymorphisms reported in gnomAD with a population frequency >1% and called as mutated in at least 75% of cells with a VAF per read count between 47% and 53% according to Tapestri Insights.Patient-specific allele dropout rates were calculated for all patients except for patient 365, which did not have any heterozygous polymorphisms fulfilling the previous criteria.In this case, we used an allele dropout rate of 0.07, which is within the range measured in the other cases.We ran ∞SCITE with and without considering NOTCH1 mutations and manually curated the result of patient 3,299 carrying an RPS15 mutation due to the high allele dropout rate observed in these genes (Supplementary Fig. 2).We ran ∞SCITE for each patient combining all time points and obtained time-point-specific subclone sizes by counting the cells assigned to each subclone in each sample 102 .Only cells uniquely assigned to one subclone were considered.Cells genotyped as wild-type for all selected mutations were considered as non-tumoral cells and were removed. Single-cell RNA-seq.Data generation.scRNA-seq was performed on longitudinal samples of five patients using three different approaches: 1. Smart-seq2: full-length scRNA-seq libraries were prepared for samples of patient 63 using the Smart-seq2 protocol 103 Read alignment.Raw reads were aligned to the GRCh38 human genome with Cell Ranger (v.4.0.0), with the 'chemistry' parameter set to 'SC3Pv3' and the 'expect-cells' parameter set to 20,000 and 5,000 for cell-hashed and non-hashed libraries, respectively.The remaining parameters for cell-hashed libraries were specified as described in the 'Feature Barcode Analysis' pipeline of Cell Ranger.For Smart-seq2 libraries, alignment and quantification was performed using zUMIs (v.9.4e) 105 . Demultiplexing of hashtag oligonucleotides.Expression matrices were imported into R (v.4.0.4) with the 'Read10X' function from Seurat (v.4.0.3) 106 .HTO counts were normalized with a centered log-ratio transformation applied across features.Each cell barcode was assigned to a specific time point of the disease with the function 'HTODemux' (positive.quantile= 0.99) of Seurat.Barcodes that were positive for two or more time points were labeled as doublets and discarded.Likewise, cell barcodes negative for all time points were excluded.Finally, Scrublet (v.0.2.1) 107 was run to aid in the detection of doublets. Quality control, normalization and dimensionality reduction.Cells that possessed <900 UMIs, <250 expressed genes or a mitochondrial expression >22.5% were considered as poor quality and removed.Similarly, genes expressed in three or fewer cells were filtered out.Following data normalization and correction (Seurat and NormalizeData), we performed PCA (Seurat, RunPCA) using the scaled expression (Seurat and ScaleData) of the top 2,000 highly variable genes (Seurat: FindVariableFeatures, selection.method= VST).For Smart-seq2 data, we filtered out cells with <150,000 counts, <550 expressed genes or mitochondrial expression >18%.Cells with more than 700,000 counts or 3,750 detected genes were excluded.Similarly, genes expressed in three or fewer cells were filtered out.To separate neoplastic cells from the microenvironment, we corrected the top 30 principal components (PCs) for sample-specific variation using Harmony (v.1.0) 108, as implemented in the RunHarmony (group.by.vars = sample) function (SeuratWrappers package, v.0.3.0).Subsequently, these 30 corrected PCs were used to embed cells in a UMAP (Seurat, RunUMAP) and in a 20-nearest neighbors graph (Seurat, FindNeighbors) for visualization and clustering, respectively.Following Louvain clustering (Seurat, FindClusters, resolution = 0.1), we focused our downstream analyses only on tumor B cells (CD79A) due to the low number of microenvironment cells. Dealing with confounders.We observed batch effects between 10x Genomics experiments.To avoid batch effects within samples of the same patient, we focused on the BCLLATLAS_10 experiment for patients 12, 19 and 3,299.Conversely, as we did not obtain a clear signal-to-noise separation in the HTO demultiplexing of case 365, we analyzed the cells obtained with BCLLATLAS_29.We also found some cell neighborhoods that harbored a high percentage of mitochondrial expression and a low number of detected genes.In such cases, we were more stringent with the thresholds or fetched and eliminated these clusters with FindClusters.We also excluded some clusters of doublets that expressed markers of microenvironment cells (erythroblasts, T cells or natural killer cells).Finally, for patient 3,299 in which one sample was obtained from peripheral blood (PB), whereas the others were obtained from bone marrow (BM), we focused solely on the BM samples to avoid misinterpretations.For patient 365, the CLL and RT time points were sampled from PB and lymph nodes, respectively.As the same RT sample profiled with bulk RNA-seq clustered with other RT samples from PB, we analyzed them jointly.After all the filtering, we recomputed the highly variable genes and PCAs.To avoid overcorrection, we used the top 20 PCs as input to RunUMAP and FindNeighbors, without rerunning Harmony. Clustering and annotation.Louvain clustering was performed with the FindClusters function, adjusting the resolution parameter for each patient independently. To annotate each cluster, we ran a 'one-versus-all' DEA for each cluster (Seurat, FindAllMarkers, Wilcoxon rank-sum test), keeping only upregulated genes with a log 2 FC > 0.3 and a Bonferroni-adjusted P value <0.001.If markers were specific to a subset of the cluster, we further stratified it with the FindSubCluster function. On the contrary, if two clusters possessed similar markers, we merged them.The CellCycleScoring function was used to identify clusters of cycling cells. DEA and GSEA.We conducted a DEA between RT and CLL clusters of each patient independently, merging cells from all time points (Seurat, FindMarkers, logfc.threshold= 0, only.pos= FALSE, Wilcoxon rank-sum test).To find finer-grained gene expression changes, only nonproliferative clusters were considered.Genes with a Bonferroni-adjusted P value <0.05 were considered as significant.The resulting list of genes (sorted by decreasing log 2 FC) was used as input to the 'gseGO' function of clusterProfiler (v.3.18.1, parameters: ont = 'BP' , OrgDB = org.Hs.eg.db, keyType = 'SYMBOL' , minGSSize = 10, maxGSSize = 250, seed = TRUE).We then removed redundancy in the output list of GO terms with the 'simplify' function (cutoff of 0.75) and filtered out GO terms with an adjusted P value <0.05.To convert the expression of specific GO terms of interest into a cell-specific score, we utilized the AddModuleScore function from Seurat. CNA inference from scRNA-seq data.For each patient separately, we ran inferCNV (v.1.11.1) integrating all samples together.We used CLL cells as reference because (1) we aimed to identify CNAs acquired at RT and (2) CLL had flat copy number profiles in virtually all chromosomes according to WGS.CLL cells were downsampled to the number of RT cells.We initialized an 'infercnv' object (CreateInfercnvObject) using the raw expression counts and the gene-ordering file https://data.broadinstitute.org/Trinity/CTAT/cnv/gencode_v21_gen_pos.complete.txt.CNAs were predicted (infercnv, run, HMM = FALSE, denoise = FALSE) setting the cutoff parameter to 1 and 0.1 for Smart-seq2 and 10x data, respectively.We customized the plotting with the plot_cnv function. Analysis of an external scRNA-seq dataset.We downloaded the expression matrices and metadata of the dataset from Penter et al. 43 with the GEOquery (v.2.62.2) (Gene Expression Omnibus identifier GSE165087), created a single Seurat object with all cells from all samples and filtered poor-quality cells as specified in the original publication 43 .Dimensionality reduction, DEA, GSEA and gene signature scoring were performed as described above. Cellular respiration.Cryopreserved cells were resuspended on RPMI-1640 (Gibco, cat.no.21875034) with 10% FBS (Gibco, cat.no.10270-106) and 1% Glutamax (Gibco, cat.no.35050-061) at a concentration of 3 million cells ml -1 .After 1 h of incubation at 37 °C, cellular respiration was performed using O 2 k-respirometers (Oroboros Instruments).Two milliliters of cell suspension were added in each respirometer chamber.Cellular respiration was performed at 37 °C at a stirrer speed of 750 r.p.m.Respiratory control was studied by sequential determination of routine respiration (oxygen consumption in living cells resuspended on RPMI-1640 with 10% FBS and 1% Glutamax), oligomycin-inhibited leak respiration (2 µl ml -1 , Sigma-Aldrich, cat.no.O4876, CAS, 1404-19-9), uncoupler-stimulated ETC measured by the sequential titration of the ionophore carbonyl cyanide m-chlorophenyl hydrazone (Sigma-Aldrich, cat.no.C2759, CAS, 555-60-2) and residual oxygen consumption after inhibition of the electron transfer system by the addition into the chamber of rotenone (0.5 µM, Sigma-Aldrich, cat.no.R8875, CAS, 83-79-4) and antimycin A (2.5 µM, Sigma-Aldrich, cat.no.A8674, CAS, 1397-94-0).Data acquisition and real-time analysis were performed using the software DatLab 7.4 (Oroboros Instruments).Automatic instrumental background corrections were applied for oxygen consumption by the polarographic oxygen sensor and oxygen diffusion into the chamber 109 .The same experimental workflow was used to study cellular respiration in CLL and RT cells after 1 h of treatment with IACS-010759 (Selleckchem, cat.no.S8731, CAS, 1570496-34-2) at 100 nM. Calcium flux analysis.Cryopreserved cells were resuspended on RPMI-1640 medium with 10% FBS, 1% Glutamax and 5% penicillin (10,000 IU ml -1) / streptomycin (10 mg ml -1 ) (Thermo Fisher, cat.no.S8731) at 10 6 cells ml -1 .After 6 h of incubation at 37 °C and 5% CO 2 , cells were centrifuged and resuspended on RPMI-1640 with 4 µM Indo-1 AM (Thermo Fisher, cat.no.I1223) and 0.08% Pluronic F-127 (Thermo Fisher, cat.no.P3000MP) for 30 min at 37 °C and 5% CO 2 .Cells were subsequently labeled for 20 min at room temperature with surface marker antibodies CD19 (Super Bright 600; Invitrogen, cat.no.63-0198-42) and CD5 (PE-Cy5; BD Biosciences, cat.no.555354) for the identification of tumoral cells (CD19 + CD5 + ).Next, cells were resuspended on RPMI-1640 before flow cytometry acquisition.Basal calcium was measured during 1 min before stimulation, then cells were incubated during 2 min at 37 °C with or without 10 µg ml -1 anti-human F(ab′)2 IgM (Southern Biotech, cat.no.2022-01) and 3.3 mM H 2 O 2 (Sigma-Aldrich, cat.no.H1009).Finally, 2 µM 4-hydroxytamoxifen (4-OHT) (Sigma-Aldrich, cat.no.H6278) was added to all conditions before continue recording for up to 8 min.Intracellular Ca 2+ release was measured on LSRFortessa (BD Biosciences) using BD FACSDiva software (v.8) by exciting with ultraviolet laser (355 nm) and appropriate filters: Indo-1 violet (450/50 nm) and Indo-1 blue (530/30 nm).Bound (Indo-1 violet) and unbound (Indo-1 blue) ratiometric was calculated with FlowJo software (v.10).Gating analysis was as follows: cell identification in FSC-A versus SSC-A plot, singlet identification in FSC-A versus FCS-H plot, tumoral cells (CD19 + CD5 + ) in CD19 (Super Bright 600) versus CD5 (PE-Cy5) plot and Ca 2+ release in time versus Indo-1 violet/Indo-1 blue plot using a kinetics tool.Optimized dilutions for the antibodies were 1:3 for CD19 and 1:10 for CD5. Cell growth assays.Cryopreserved cells were resuspended on PBS at a concentration of 10 7 cells ml -1 and labeled with 0.5 µM CFSE Cell Tracer (Thermo Fisher, cat.no.C34554) for 10 min.Cells were centrifuged and resuspended on enriched RPMI-1640 medium with 1% Glutamax, 15% FBS, 1× insulin-transferrin-selenium (Merk, cat.no.I3146), 10 mM HEPES (Fisher Scientific, cat.no.BP299), 50 µM 2-mercaptoethanol (Gibco, cat.no.21985-023), 1× Non-Essential Amino Acids (Gibco, cat.no.11140-050), 1 mM sodium pyruvate (Gibco, cat.no.11360-070) and 50 µg ml -1 gentamicin (Gibco, cat.no.15710-064) at a concentration of 10 6 cells ml -1 supplemented with 0.2 µM CpG DNA TLR9 ligand (ODN2006-TL9; InvivoGen, cat.no.TLRL-2006) and 15 ng ml -1 recombinant human IL-15 (R&D Systems, cat.no.247-ILB-025) 110 .When indicated, cells were treated for 72 h with 100 nM IACS-010759.Cells were labeled for 20 min at room temperature with surface marker antibodies CD19 (Super Bright 600), CD5 (PE-Cy5) and annexin V (Life Technologies, cat. no.A35122) before acquisition in a LSRFortessa (BD Biosciences) using the BD FACSDiva software (v.8) and analyzed using FlowJo (v.10).Gating analysis for divided cells was as follows: cell identification in FSC-A versus SSC-A plot, singlet identification in FSC-A versus FCS-H plot, alive cells in annexin V (PacB) versus SSC-A plot, tumoral cells (CD19 + CD5 + ) in CD19 (Super Bright 600) versus CD5 (PE-Cy5) plot and proliferating cells in the CFSE histogram.Optimized dilutions for the antibodies were 1:3 for CD19, 1:10 for CD5 and 1:3 for annexin V. Reporting summary.Further information on research design is available in the Nature Research Reporting Summary linked to this article.The plot shows the percentage of samples with gains (up) and losses (down).Among recurrent alterations found either in CLL or RT samples (n ≥ 5), deletions of 9p (PTPRD and CDKN2A/B) and deletions of 15q (MGA) were enriched in RT whereas deletions of ATM (11q), TP53 (17p), and 13q14 were found at similar frequencies in CLL and RT.e. Oncoprint of putative driver alterations.Samples, grouped by patient (patient id at the top), are represented by columns while genes in rows.Novel drivers in RT are labeled in blue.Genes are grouped according to their biological function or if they were previously described as potential driver genes in CLL and/or mature B cell lymphomas.Metadata including the type of therapy before RT, number of treatment lines before each sample, the spatial/longitudinal nature of the CLL/RT samples analyzed, IGHV mutational status, and diagnosis is detailed in the upper rows.In the main plot, mutations (SNVs and indels) are depicted with horizontal rectangles, CNAs using the background color of each cell, and SVs with vertical rectangles.The transparency of the color of mutations and CNAs indicates the cancer cell fraction (CCF).For patients lacking the germline sample (patient id indicated in gray), the CCF of the alterations could not be inferred and a CCF of 100% was used for illustrative purposes. ",
"section_name": "Detection of differential epigenetic regions and RT-specific changes.",
"section_num": null
}
] |
[
{
"section_content": "The authors thank the Hematopathology Collection registered at the Biobank of Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) and the Biobank HUB-ICO-IDIBELL (PT20/00171) for sample procurement, S. Martín, F. Arenas, the Genomics Core Facility of the IDIBAPS, CNAG Sequencing Unit, Mission Bio, Omniscope and Barcelona Supercomputing Center for the technical support and the computer resources at MareNostrum4 (RES activity, BCV-2018-3-0001).This study was supported by the la Caixa Foundation (CLLEvolution-LCF/PR/HR17/52150017, Health Research 2017 Program HR17-00221, to E.C.), the European Research Council under the European Union's Horizon 2020 Research and Innovation Program (810287, BCLLatlas, to E.C., J.I.M.-S., H.H. and I.G.), the Instituto de Salud Carlos III and the European Regional Development Fund Una Manera de Hacer Europa (PMP15/00007 to E.C. and RTI2018-094584-B-I00 to D.C.), the American Association for Cancer Research (2021 AACR-Amgen Fellowship in Clinical/Translational Cancer Research, 21-40-11-NADE to F.N.), the European Hematology Association (EHA Junior Research Grant 2021, RG-202012-00245 to F.N.), the Lady Tata Memorial Trust (International Award for Research in Leukaemia 2021-2022, LADY_TATA_21_3223 to F.N.), the Generalitat de Catalunya Suport Grups de Recerca AGAUR (2017-SGR-1142 to E.C., 2017-SGR-736 to J.I.M.-S.and 2017-SGR-1009 to D.C.), the Accelerator award CRUK/AIRC/ AECC joint funder partnership (AECC_AA17_SUBERO to J.I.M.-S. ), the Fundació La Marató de TV3 (201924-30 to J.I.M.-S.), the Centro de Investigación Biomédica en Red Cáncer (CIBERONC; CB16/12/00225, CB16/12/00334, CB16/12/00236), the Ministerio de Ciencia e Innovación (PID2020-117185RB-I00 to X.S.P.), the Fundación Asociación Española Contra el Cáncer (FUNCAR-PRYGN211258SUÁR to X.S.P.), the Associazione Italiana per la Ricerca sul Cancro Foundation (AIRC 5 × 1,000 no.21198 to G.G.) and the CERCA Programme/Generalitat de Catalunya.H.P.-A. is a recipient of a predoctoral fellowship from the Spanish Ministry of Science, Innovation and Universities (FPU19/03110).A.D.-N. is supported by the Department of Education of the Basque Government (PRE_2017_1_0100).E.C. is an Academia Researcher of the Institució Catalana de Recerca i Estudis Avançats of the Generalitat de Catalunya.This work was partially developed at the Center Esther Koplowitz (Barcelona, Spain). ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "Sequencing data are available from the European Genome-phenome Archive (http://www.ebi.ac.uk/ega/) under accession no.EGAS00001006327.scRNA-seq expression matrices, Seurat objects and corresponding metadata are available at Zenodo (https://doi.org/10.5281/zenodo.6631966). R markdown notebooks used for mutational signature, bulk RNA-seq, H3K27ac and ATAC-seq analyses can be found at https://github.com/ferrannadeu/RichterTransformation.R markdown notebooks to reproduce the scRNA-seq analyses can be accessed at https://github.com/massonix/richter_transformation.Code to normalize DNA methylation data can be found at https://github.com/Duran-FerrerM/DNAmeth_arrays.Code to calculate the tumor cell content, CLL epitypes and epiCMIT from DNA methylation data can be found at https://github.com/Duran-FerrerM/Pan-B-cell-methylome. Fig. 4 | See next page for caption.NATuRE MEDICINE | www.nature.com/naturemedicineExtended Data Fig. 7 | See next page for caption.NATuRE MEDICINE | www.nature.com/naturemedicine ",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "Sequencing data are available from the European Genome-phenome Archive (http://www.ebi.ac.uk/ega/) under accession no.EGAS00001006327.scRNA-seq expression matrices, Seurat objects and corresponding metadata are available at Zenodo (https://doi.org/10.5281/zenodo.6631966). ",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "R markdown notebooks used for mutational signature, bulk RNA-seq, H3K27ac and ATAC-seq analyses can be found at https://github.com/ferrannadeu/RichterTransformation.R markdown notebooks to reproduce the scRNA-seq analyses can be accessed at https://github.com/massonix/richter_transformation.Code to normalize DNA methylation data can be found at https://github.com/Duran-FerrerM/DNAmeth_arrays.Code to calculate the tumor cell content, CLL epitypes and epiCMIT from DNA methylation data can be found at https://github.com/Duran-FerrerM/Pan-B-cell-methylome. ",
"section_name": "Code availability",
"section_num": null
},
{
"section_content": "Fig. 4 | See next page for caption.NATuRE MEDICINE | www.nature.com/naturemedicineExtended Data Fig. 7 | See next page for caption.NATuRE MEDICINE | www.nature.com/naturemedicine ",
"section_name": "Extended Data",
"section_num": null
},
{
"section_content": "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/. F.N. has received honoraria from Janssen and AbbVie for speaking at educational activities.J.L.M. is an employee of Omniscope.X.S.P. is cofounder of and holds an equity stake in DREAMgenics.H.H. is cofounder of Omniscope and consultant to MiRXES.E.C. has been a consultant for Takeda, NanoString, AbbVie and Illumina; has received honoraria from Janssen, EUSPharma and Roche for speaking at educational activities; and is an inventor on a Lymphoma and Leukemia Molecular Profiling Project patent 'Method for subtyping lymphoma subtypes by means of expression profiling' (PCT/US2014/64161) not related to this project.The remaining authors declare no competing interests. Extended data is available for this paper at https://doi.org/10.1038/s41591-022-01927-8. The online version contains supplementary material available at https://doi.org/10.1038/s41591-022-01927-8. Extended Data Fig. 3 | Complex genomic rearrangements affecting driver genes.a. Deletions in chr12 identified in four cases with the minimal deleted region affecting CDKN1B, which expression in CLL and RT sample pairs is shown on the right.The case carrying the deletion at time of RT is labeled in the boxplot.b.Reciprocal translocation juxtaposing CDK6 next to IGKJ5 in patient 4687.c.Deletion in chr1 affecting two cases with the minimal deleted region targeting ARID4B.Its expression in CLL and RT sample pairs is shown in the boxplot on the right.d.Reciprocal translocations truncating CREBBP and CIITA in the RT sample of patient 12. e.Expression levels of known and novel RT-driver genes in CLL and RT paired samples.Cases carrying deletions/ mutations at time of RT are labeled.f-j.Complex genomic rearrangements affecting driver genes in five selected RT samples.The circos plots show the SVs (inner links) and CNAs (middle circle) found in each sample.SVs are colored based on whether they are part of a complex event, while CNAs are painted according to their type.Chromosome-specific plots on the right show the main chromosomes affected by complex events targeting driver genes (annotated at the bottom).In these chromosome-specific plots, the color of both CNAs and SVs indicates their type.For patient 12 (f), the expression levels of three genes affected by simple (TRAF3) and complex (SPEN and TNFRS14) chromosomal alterations are shown.For patient 4675 (j), the partner of the translocations found in chr3 and chr8 are not specified for simplicity due to the high number of clustered structural events.All boxplots: center line, median; box limits, upper/lower quartiles; whiskers, 1.5×interquartile range; points, individual samples.All p values are from two-sided T tests. Extended Data Fig. 4 | Extraction and assignment of mutational signatures.a-d.Signatures extracted by the Hierarchical Dirichlet Process (HDP) (a), SignatureAnalyzer (b), SigProfiler (c), and sigfit (d).COSMIC signatures needed to reconstruct the extracted signatures are shown together with their contribution (in percentage).The cosine similarities between the extracted and reconstructed signatures are shown in brackets.e. Workflow of the mutational signature analysis.f.The 96-mutation profile of the RT sample of patient 839 (time point 2), which had marked evidence of APOBEC activity (SBS2 and SBS13).g.Comparison of the SBS-ganciclovir extracted by HDP and SignatureAnalyzer.Based on the high cosine similarity (0.996), we considered that both signatures represented the same mutational process and selected the one extracted by HDP for downstream analyses.h.Comparison of the SBS-ganciclovir extracted by HDP and the ganciclovir signature reported by de Kanter et al. 35 .i. Comparison of the SBS-RT extracted by HDP and SignatureAnalyzer.Based on the high cosine similarity (0.941), we considered that both signatures represented the same mutational process and selected the one extracted by HDP for downstream analyses.j.Pairwise comparisons of the SBS-RT with known signatures from COSMIC and Kucab et al. 33 .k. Decomposition of the SBS-RT in \"n\" known signatures using an expectation maximization approach.The low cosine similarity (<0.85) between SBS-RT and the best reconstituted signature obtained using any combination of known signatures suggests that SBS-RT represents a novel mutational signature. Extended Data Fig. 7 | scRNA-seq characterization of CLL and RT.a-d.UMAP visualization of tumor cells from all time points colored by annotation and tissue of origin.hi, high; lo, low; PB, peripheral blood; LN, lymph node; BM, bone marrow (left).Dot plot with the expression of key markers in each cluster.Color and size represent scaled mean expression and proportion of cells expressing each marker gene, respectively (middle-left).Violin plots showing the cell-cycle phase scores (S and G-to-M) for each cluster of cells (middle-right).UMAP visualization split by time point (right).'n' refers to the total number of cells in that time point, and the percentage refers to the proportion of cells within RT clusters.e-i.Time point-specific UMAP visualizations for each case.RT seed cells are depicted in rose and with an increased size.j.UMAP visualization of case CLL9 from Penter et al. 43 43 .l. GSEA between RT and CLL cells of patient CLL9 from Penter et al. 43 . ",
"section_name": "© The Author(s) 2022",
"section_num": null
},
{
"section_content": "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": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "© The Author(s) 2022",
"section_num": null
},
{
"section_content": "F.N. has received honoraria from Janssen and AbbVie for speaking at educational activities.J.L.M. is an employee of Omniscope.X.S.P. is cofounder of and holds an equity stake in DREAMgenics.H.H. is cofounder of Omniscope and consultant to MiRXES.E.C. has been a consultant for Takeda, NanoString, AbbVie and Illumina; has received honoraria from Janssen, EUSPharma and Roche for speaking at educational activities; and is an inventor on a Lymphoma and Leukemia Molecular Profiling Project patent 'Method for subtyping lymphoma subtypes by means of expression profiling' (PCT/US2014/64161) not related to this project.The remaining authors declare no competing interests. ",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Extended data is available for this paper at https://doi.org/10.1038/s41591-022-01927-8. ",
"section_name": "Additional information",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https://doi.org/10.1038/s41591-022-01927-8. ",
"section_name": "Supplementary information",
"section_num": null
},
{
"section_content": "",
"section_name": "Articles",
"section_num": null
},
{
"section_content": "Extended Data Fig. 3 | Complex genomic rearrangements affecting driver genes.a. Deletions in chr12 identified in four cases with the minimal deleted region affecting CDKN1B, which expression in CLL and RT sample pairs is shown on the right.The case carrying the deletion at time of RT is labeled in the boxplot.b.Reciprocal translocation juxtaposing CDK6 next to IGKJ5 in patient 4687.c.Deletion in chr1 affecting two cases with the minimal deleted region targeting ARID4B.Its expression in CLL and RT sample pairs is shown in the boxplot on the right.d.Reciprocal translocations truncating CREBBP and CIITA in the RT sample of patient 12. e.Expression levels of known and novel RT-driver genes in CLL and RT paired samples.Cases carrying deletions/ mutations at time of RT are labeled.f-j.Complex genomic rearrangements affecting driver genes in five selected RT samples.The circos plots show the SVs (inner links) and CNAs (middle circle) found in each sample.SVs are colored based on whether they are part of a complex event, while CNAs are painted according to their type.Chromosome-specific plots on the right show the main chromosomes affected by complex events targeting driver genes (annotated at the bottom).In these chromosome-specific plots, the color of both CNAs and SVs indicates their type.For patient 12 (f), the expression levels of three genes affected by simple (TRAF3) and complex (SPEN and TNFRS14) chromosomal alterations are shown.For patient 4675 (j), the partner of the translocations found in chr3 and chr8 are not specified for simplicity due to the high number of clustered structural events.All boxplots: center line, median; box limits, upper/lower quartiles; whiskers, 1.5×interquartile range; points, individual samples.All p values are from two-sided T tests. ",
"section_name": "NATuRE MEDiCiNE",
"section_num": null
},
{
"section_content": "Extended Data Fig. 4 | Extraction and assignment of mutational signatures.a-d.Signatures extracted by the Hierarchical Dirichlet Process (HDP) (a), SignatureAnalyzer (b), SigProfiler (c), and sigfit (d).COSMIC signatures needed to reconstruct the extracted signatures are shown together with their contribution (in percentage).The cosine similarities between the extracted and reconstructed signatures are shown in brackets.e. Workflow of the mutational signature analysis.f.The 96-mutation profile of the RT sample of patient 839 (time point 2), which had marked evidence of APOBEC activity (SBS2 and SBS13).g.Comparison of the SBS-ganciclovir extracted by HDP and SignatureAnalyzer.Based on the high cosine similarity (0.996), we considered that both signatures represented the same mutational process and selected the one extracted by HDP for downstream analyses.h.Comparison of the SBS-ganciclovir extracted by HDP and the ganciclovir signature reported by de Kanter et al. 35 .i. Comparison of the SBS-RT extracted by HDP and SignatureAnalyzer.Based on the high cosine similarity (0.941), we considered that both signatures represented the same mutational process and selected the one extracted by HDP for downstream analyses.j.Pairwise comparisons of the SBS-RT with known signatures from COSMIC and Kucab et al. 33 .k. Decomposition of the SBS-RT in \"n\" known signatures using an expectation maximization approach.The low cosine similarity (<0.85) between SBS-RT and the best reconstituted signature obtained using any combination of known signatures suggests that SBS-RT represents a novel mutational signature. ",
"section_name": "NATuRE MEDiCiNE",
"section_num": null
},
{
"section_content": "Extended Data Fig. 7 | scRNA-seq characterization of CLL and RT.a-d.UMAP visualization of tumor cells from all time points colored by annotation and tissue of origin.hi, high; lo, low; PB, peripheral blood; LN, lymph node; BM, bone marrow (left).Dot plot with the expression of key markers in each cluster.Color and size represent scaled mean expression and proportion of cells expressing each marker gene, respectively (middle-left).Violin plots showing the cell-cycle phase scores (S and G-to-M) for each cluster of cells (middle-right).UMAP visualization split by time point (right).'n' refers to the total number of cells in that time point, and the percentage refers to the proportion of cells within RT clusters.e-i.Time point-specific UMAP visualizations for each case.RT seed cells are depicted in rose and with an increased size.j.UMAP visualization of case CLL9 from Penter et al. 43 43 .l. GSEA between RT and CLL cells of patient CLL9 from Penter et al. 43 . ",
"section_name": "NATuRE MEDiCiNE",
"section_num": null
}
] |
10.1371/journal.pone.0127209
|
Avoiding False Positive Antigen Detection by Flow Cytometry on Blood Cell Derived Microparticles: The Importance of an Appropriate Negative Control
|
Microparticles (MPs), also called microvesicles (MVs) are plasma membrane-derived fragments with sizes ranging from 0.1 to 1μm. Characterization of these MPs is often performed by flow cytometry but there is no consensus on the appropriate negative control to use that can lead to false positive results.We analyzed MPs from platelets, B-cells, T-cells, NK-cells, monocytes, and chronic lymphocytic leukemia (CLL) B-cells. Cells were purified by positive magnetic-separation and cultured for 48h. Cells and MPs were characterized using the following monoclonal antibodies (CD19,20 for B-cells, CD3,8,5,27 for T-cells, CD16,56 for NK-cells, CD14,11c for monocytes, CD41,61 for platelets). Isolated MPs were stained with annexin-V-FITC and gated between 300nm and 900nm. The latex bead technique was then performed for easy detection of MPs. Samples were analyzed by Transmission (TEM) and Scanning Electron microscopy (SEM).Annexin-V positive events within a gate of 300-900nm were detected and defined as MPs. Our results confirmed that the characteristic antigens CD41/CD61 were found on platelet-derived-MPs validating our technique. However, for MPs derived from other cell types, we were unable to detect any antigen, although they were clearly expressed on the MP-producing cells in the contrary of several data published in the literature. Using the latex bead technique, we confirmed detection of CD41,61. However, the apparent expression of other antigens (already deemed positive in several studies) was determined to be false positive, indicated by negative controls (same labeling was used on MPs from different origins).We observed that mother cell antigens were not always detected on corresponding MPs by direct flow cytometry or latex bead cytometry. Our data highlighted that false positive results could be generated due to antibody aspecificity and that phenotypic characterization of MPs is a difficult field requiring the use of several negative controls.
|
[
{
"section_content": "We analyzed MPs from platelets, B-cells, T-cells, NK-cells, monocytes, and chronic lymphocytic leukemia (CLL) B-cells.Cells were purified by positive magnetic-separation and cultured for 48h.Cells and MPs were characterized using the following monoclonal antibodies (CD19,20 for B-cells, CD3,8,5,27 for T-cells, CD16,56 for NK-cells, CD14,11c for monocytes, CD41,61 for platelets).Isolated MPs were stained with annexin-V-FITC and gated between 300nm and 900nm.The latex bead technique was then performed for easy detection of MPs.Samples were analyzed by Transmission (TEM) and Scanning Electron microscopy (SEM). ",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "Annexin-V positive events within a gate of 300-900nm were detected and defined as MPs.Our results confirmed that the characteristic antigens CD41/CD61 were found on plateletderived-MPs validating our technique.However, for MPs derived from other cell types, we were unable to detect any antigen, although they were clearly expressed on the MP-producing cells in the contrary of several data published in the literature.Using the latex bead technique, we confirmed detection of CD41,61.However, the apparent expression of other ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "In recent years, a large number of publications have established that cells are able to produce ''extracellular vesicles\" (EVs), which are important mediators of physiological processes in normal and pathological cells (e.g., cell growth, activation, proliferation, apoptosis, senescence) [1;2].EVs principally include three populations distinguishable by size, composition and biogenesis: exosomes (50-100 nm in diameter), microparticles (100 nm to 1 μm) and apoptotic bodies (AB; 1 μm to 4 μm) [3].In this study, we focused on microparticles (MPs), also called microvesicles (MVs) by some authors.These particles are released into the extracellular space by outward budding and fission of the plasma membrane [4][5][6].The release of vesicles is efficiently induced upon cellular activation or apoptosis and the subsequent increase of intracellular Ca2+.These MPs contain proteins and nucleic acids, including cytoplasmic and membrane proteins [7], mRNAs [8;9], microRNAs (miRNAs) [10][11][12], non-coding RNAs (ncRNAs) [13], and DNA [14][15][16][17].All of these elements can be delivered to other cells by different mechanisms [4;18].MPs normally feature antigens from parental cells and phosphatidylserine (PS), which can be detected by annexin-V staining [19;20].However, some observations also suggest the existence of MPs without PS externalization [21][22][23][24][25]. The characterization of MPs is most often performed by flow cytometry, which is considered the gold standard technique used in 75% of MP publications.Lacroix et al defined an accurate MP gate between 0.3 and 1 μm as the best compromise between good resolution and a level of background noise that does not impede cytometer performance [26].Over the years, other techniques have been applied to improve the study of MPs such as electron microscopy, ELISA, nanoparticle tracking analysis, and atomic force microscopy [27]. The field of MP study is rapidly expanding.It has been already shown that MPs in body fluids could be used as prognostic markers for pathologies that include cardiovascular diseases, inflammation, sepsis, lupus, HIV, and several cancers [28][29][30][31].MPs also have significant potential for clinical applications, especially in brain cancer, where EVs have been used as delivery vehicle to transport therapeutic molecules [32][33][34]. However, some discrepancies exist in literature concerning phenotypic characterization of MPs.Ghosh et al [35] and Macey et al [36] were able to detect some CD19+ B lymphocyte-derived MPs, Blanchard et al [37] showed CD3+ T lymphocyte-derived MVs while Miguet et al [38] demonstrated by proteomic study that these antigens were not found in vesicles.Blanchard et al highlighted also that CD28, CD40L and CD45 were not found on MVs derived from T lymphocytes despite these antigens were clearly detected in the original cells [37].In addition, since MP analysis by flow cytometry is quite difficult due to their small size, several authors [37;39;40] used technique based on latex beads with different protocols.These beads can generate non-specific staining depending on the choice of antibody, or saturation methods and thus false positive results.In the present paper, we demonstrated that several results published in the literature are more than probably wrong due to the use of inappropriate controls.The purpose of this study was thus to clarify antigen detection on MPs from blood cells and to propose new negative control in MP analysis by flow cytometry to avoid false positive results. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "This study has been approved by the Bordet Institute Ethics Committee and conducted according to the principles expressed in the Declaration of Helsinki.All samples were collected after written informed consent. ",
"section_name": "Ethics statement",
"section_num": null
},
{
"section_content": "Peripheral blood mononuclear cells (PBMC) were obtained from healthy donor buffy coat (provided by the \"service francophone du sang de la croix rouge de Belgique\").PBMC were isolated by Ficoll-Hypaque gradient centrifugation as previously described [41].B-cells, T-cells, NK-cells, and monocytes were purified by positive selection, using CD19, CD3, CD56, and CD14 microbeads, respectively (Macs Miltenyi Biotec, Leiden, the Netherlands).The purity of cells was from 97.46 to 99% after immune magnetic enrichment (data not shown).Platelets were obtained from expired single-donor platelet apheresis units.They were collected by the \"service francophone du sang de la croix rouge de Belgique\".Chronic Lymphocytic Leukemia (CLL) samples were obtained from CLL patients with informed written consent.The normal purified cells and CLL B-cells were cultured at densities between 4-70 x 10 6 cells/ml (according to cell type) in 0.2 μm filtered RPMI 1640 (Lonza, Basel, Switzerland).A serum-free medium was used to avoid contamination by Fetal Bovine Serum (FBS)-derived vesicles [42].We also used bone marrow mesenchymal stromal cells (BM-MSCs) to include a non-hematopoietic negative control.BM-MSCs were harvested from the sternum or iliac crest of healthy volunteers and were isolated by the classical adhesion method, as previously described [43].BM-MSCs were plated at 1000 cells/cm 2 and cultures were used at a subconfluent state (2 x 10 6 cells).BM-MSCs were cultured in a T-175 flask with serum-free DMEM (Dulbecco's modified Eagle medium-low glucose) filtered through a 0.2 μm filter.Cells were incubated at 37°C in a 5% CO 2 humidified atmosphere.With the exception of platelets, all samples were cultured for 2 days prior to MP isolation.Cell viability was analyzed by Trypan blue exclusion assay and confirmed by Annexin V/7AAD labeling (S1 Table ). ",
"section_name": "Biological samples and cell culture",
"section_num": null
},
{
"section_content": "MPs were prepared from the supernatant of several cell cultures (healthy purified cells, CLL cells, and BM-MSCs).Although a standardized centrifugation protocol has not been established, a majority of authors have used a 20,000 x g centrifugation for MP recovery, while a 100,000 to 200,000 x g centrifugation is generally necessary to isolate exosomes [26;44-46].Cell-free supernatant (1ml of blood cell culture and 50ml of MSC culture) was obtained by 2 successive centrifugations at 300 x g for 10 minutes (for platelet samples, centrifugation at 450 x g for 15 minutes was used to remove a majority of platelets).The supernatants were then subjected to 20,000 x g centrifugation for 1 h at 4°C (Ultracentrifuge MX 120+, Swinging Bucket rotor S50-ST, k-factor 77).The MP pellet was washed using 0.2 μm filtered phosphate buffered saline (PBS) and MPs were again centrifuged for 1 h at 20, 000 x g.The MP pellet was finally reconstituted in 150 μl of PBS and stored at -80°C.To ensure that the freezing step do not affect the labeling results, we performed a staining comparison between the samples, before and after the congelation (data not shown). ",
"section_name": "Microparticle isolation",
"section_num": null
},
{
"section_content": "Because unfiltered buffers and antibodies have been shown to contain interfering elements that can give false positive signals in cytometry [47][48][49][50][51], all products used here were filtered through 0.2 μm filters (VWR, Leuven, Belgium).The following monoclonal antibodies were used: CD19-PE (LT19), CD3-PE(BW264/56), and CD14-PE(TÜK4), CD61-PE(Y2/51) (Macs Miltenyi).CD19-PC5(HIB19), CD20-PE(2H7), CD11c-PE(B-ly6) (BD Pharmingen, Erembodegen, Belgium), CD16-PE(3G8), CD41-PE(P2), CD56-PC5(HLDA6), CD5-PE(BL1a)(Beckman Coulter, Marseille, France), CD8-PC5(DK25) (Dako, Heverlee, Belgium) and CD27-Percp (0323) (Biolegend, San Diego, CA, USA).Annexin-V (Invitrogen) and 7-AAD (BD Pharmingen) were applied to detect apoptotic bodies.Appropriate PE, PE-Cy5, APC isotypes were used as negative controls, all from BD Pharmingen.All the concentrations/clones/origins were reported in S2 Table .Each couple of antibodies were tested in PBS alone to prove their perfect matching, the geometric means were similar between specific antibodies and their isotype control, for all the tested antibodies (data not shown). ",
"section_name": "Antibodies and other reagents",
"section_num": null
},
{
"section_content": "All analyses were performed on a Navios cytometer (Beckman Coulter).The MP gate was established based on light scattering and size properties (Forward scatter-FSC W2), using Megamix Plus-FSC beads of 0.1, 0.3, 0.5, and 0.9 μm (Biocytex, Marseille, France) and defining MPs as events <1 μm.The lower detection limit was defined as a threshold above the electronic noise of the flow cytometer (0.3 μm).Annexin-V-FITC was used as a general marker for MPs [25;52].Both annexin-V positive and negative MPs were analyzed in all experiments.Cells and MPs were characterized for the expression of following antigens: CD19 and 20 for B-cells, CD3, 8, 5, and 27 for T-cells, CD16 and 56 for NK-cells, CD14 and 11c for monocytes, CD41 and 61 for platelets.A total of 10 μl of isolated MPs were stained for 15 minutes at room temperature in the dark with 10 μl of annexin-V-FITC and 10 μl of specific antibody.The samples were previously diluted with 20 μl of PBS and 50 μl specific buffer for annexin-V binding (Invitrogen).We performed serial dilutions of antibodies to determine the optimal antibody dilution and to optimize the separation of positive and negative signals.We also used different fluorochromes and isotype controls to confirm our previous results (S3 and S4 Tables).The CD3 titration on T-cells and T-cell derived MPs and CD41 on platelet derived MPs are shown on S1 and S2 Figs. Events were acquired during 2 minutes of medium flow.Lysis with 0.05% triton was used to monitor false positive signals caused by protein complex (PC); MPs, but not PCs, have been shown to be lysed by triton [53].To ensure that we analyzed MPs only, we labeled samples with annexin-V and 7-AAD to detect the possible presence of AB.The same labeling was realized without Ca 2+ binding and without 7AAD as a negative control. For cell immunophenotypic analysis, all cells were washed in PBS, suspended in 100 μl and labeled with a cell-specific antibody (10 μl) for 15 minutes in the dark.Cells were washed with PBS and a minimum of 20,000 cells were acquired.Data collected from all experiments were analyzed using FSC 3.0 (De novo analysis software, Los Angeles, CA, USA). ",
"section_name": "Cell and MP flow cytometry analysis",
"section_num": null
},
{
"section_content": "Different protocols utilizing latex beads can be found in the literature [37;54;55].We first used the method described by Wu et al and Mokarizadeh et al [39;40], but we obtained false positive results when MP-coated beads were incubated with antibodies despite the use of a blocking solution such as Bovine Serum Albumin (BSA), Fetal Calf Serum (FCS) or glycine solution, confirming the observations made by Oksvold et al. [56].Protein concentration was determined with a Nanodrop (Thermo scientific, Nanodrop 2000c) [57].A total of 15 μg (60 μg for platelets) of MPs in 100 μl of PBS were incubated with 10 μl of antibody for 15 minutes in the dark.Samples were washed with PBS and centrifuged at 20,000 x g for 1 hour at 4°C.Labeled MPs were resuspended in 1 ml of PBS and 1 μl of beads (aldehyde sulfate latex beads 4 μm-Invitrogen) was added.Samples were incubated overnight at 4°C under gentle agitation, followed by a wash step with centrifugation at 300 x g for 10 minutes.Beads were also incubated with the antibody ''washed at 20,000 x g\" (without MPs) as the negative control.Labeling of MPs with a negative marker (a marker that is not present on the cells from which the MPs were derived; for example, CD19 for CD3-derived MPs) before incubation with beads was used as a second negative control.The coated beads were resuspended in PBS before reading with a MACS-Quant analyzer (Miltenyi Biotec).All data were analyzed using FSC 3.0 (De novo analysis software, Los Angeles, CA, USA). ",
"section_name": "Characterization of MPs by the latex bead technique",
"section_num": null
},
{
"section_content": "Electron microscopy was used to study the morphology of MPs.The MP pellets obtained by centrifugation were submitted for TEM (transmission electron microscopy) and SEM (scanning electron microscopy).For TEM, 40 μl of vesicle suspension were placed on a carbon-coated EM grid, and 0.4 μl of 25% glutaraldehyde was added.Vesicles were then allowed to settle onto the grid overnight at 4°C.Grids were then blotted on filter paper and stained for 30 seconds with 2% uranyl acetate.After further blotting and drying, samples were directly observed on a Tecnai 10 TEM (FEI).Images were captured with a Veleta camera and processed with iTEM and Adobe Photoshop software.For SEM, samples were fixed overnight at 4°C in 2.5% glutaraldehyde, 0.1 M cacodylate buffer (pH 7.2); placed on a glass coverslip; and incubated overnight at 4°C to allow the vesicles to settle.Samples were then rinsed and post-fixed in 2% OsO 4 for 1 h.After serial dehydration in ethanol, the samples were critical-point dried and coated with platinum according to standard procedures.Imaging was carried out on a Tecnai FEG ESEM Quanta 200 (FEI), and images were processed with iTEM and Adobe Photoshop.We also performed immunogold labeling to confirm our data with a more sensitive technique to detect antigen on MPs.For this assays, purified MPs were fixed in paraformaldehyde 2% as previously described by C. Thery et al. [58].Grids containing the samples were blocked with PBS with 10% FBS (previously ultracentrifuged during 18h [59]).Each antibody was diluted in PBS with 5% FBS to obtain a concentration of 5μg/ml.Antibody (CD5) was incubated with MPs during 30 minutes before 3 washing.The grids were then incubated with gold-labeled secondary antibodies (goat secondary antibody to mouse IgG 10nm gold /Abcam, Cambridge, England) diluted in PBS with 2% FBS for 30 min, and then washed 3 times.The grids were then observed under electron microscope.These data were added in S3 Fig. ",
"section_name": "Transmission and scanning electron microscopy",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To characterize MPs, a Navios cytometer (Beckman Coulter, Marseille, France), an FSC optimized instrument, was used.As shown in Fig 1, the cytometer was calibrated using a standardized protocol from Biocytex.Briefly, 0.1 μm, 0.3 μm, 0.5 μm, and 0.9 μm Megamix beads-FCS plus were detected in channel FL1 (Fig 1A).With the forward scattering resolution of this cytometer, we were able to accurately distinguish the different bead sizes (Fig 1B ), which allowed to define a standardized size-related gate for MP analysis (Fig 1C).Fig 1D shows a representative scatter plot of an MP population from PBMC.We also observed that the population of smaller MPs was more significant than the population of larger MPs.Double labeling with annexin-V-FITC and CD41-PE allowed targeting of platelet derived-MPs (Fig 1E).Although apoptotic bodies (AB) were excluded by size criteria, we monitored for their possible presence by double staining with annexin V and 7AAD.As shown in Fig 1F ,AB were not detected in our samples.The presence of protein complex (PC) can also interfere with MP analysis; therefore, we used lysis with 0.05% triton to evaluate the proportion of PC, which we found was <2% in all analyzed samples (Fig 1G).Finally, MPs were analyzed by electron microscopy.MP analysis by flow cytometry and electron microscopy.Calibration of Navios cytometer using 0.1, 0.3, 0.5, and 0.9 μm beads (Biocytex).The cytometer is able to differentiate the 4 different populations of beads (A-B).We can delimit the gate in accordance with size to study MPs (0.3-0.9 μm) (C).Characteristic elongated shape of MPs repartition limited in a gate of 0.3-0.9μm.(D).Labeling of platelet derived-MPs with CD41 and annexin-V revealed a double positive population for these markers (E).Example of 7-AAD/annexin V labeling to detect the presence of apoptotic bodies (AB).(F).Absence of contamination with AB.To monitor the presence of protein complexes (PC), lysis with 0.05% triton was used.After lysis, the positive signal disappeared (G). ",
"section_name": "Detection of MPs by flow cytometry and electron microscopy",
"section_num": null
},
{
"section_content": "",
"section_name": "Scanning Electron Microscopy (SEM) shows the production of MPs by B-lymphocytes (white arrows) (H). Transmission electron microscopy (TEM) shows the structure of one MP with a characteristic lipid bilayer (white arrows) (I).",
"section_num": null
},
{
"section_content": "We first investigated the expression of several antigens on the cell surface and cell-derived MPs by direct flow cytometry (n = 5).Signals from platelet markers (CD41 and CD61) were clearly positive on both platelet-derived MPs and platelets (Fig 2A -2C).These initial results indicated that we were able to detect annexin V+/marker+ MPs.These first 2 antigens were largely described in the MP literature and were used as positive controls to initialize the setup of our experiments.However, for all other cell-derived MPs (from B-cells, T-cells, NK cells, monocytes, and CLL B-cells), the markers used were not detected, despite the fact that these antigens were highly expressed by the source cells.A representative example of some of these markers is provided in Fig 2 .For CD56, CD3, and CD19 staining, a weak positive signal was observed compared to the isotopic control (Fig 2J , 2M and2Q).However, this signal was a false positive, shown by using the same antibodies to label MPs from different origins (Fig 3).It should be noted that CD14 labeling on monocytes was quite weak, most likely due to the positive selection with anti-CD14 beads (Fig 2I).Heterogeneous expression of phosphatidylserine dependent on the cellular source of MPs was also observed.Double staining with annexin-V-FITC and specific markers was applied on four MP types (B-lymphocytes, T-lymphocytes, monocytes, NK-cells) with cross-labeling different MPs to detect false positive expression (Fig 3).Histograms of these cell derived-MPs stained with CD19-PE, CD3-PE, CD14-PE, and CD16-PE showed that non-specific labeling occurred in a majority of samples.Means of the Mean Fluorescence Intensity Ratio (MFIR) for all antibodies (studied on both annexin-V positive and negative MPs, as well as cell populations) are provided in Table 1.This study demonstrated that all antigens were clearly detected on cells but not on their corresponding MPs, with the exceptions of CD41 and CD61 for platelet-derived MPs.We confirmed the detection of CD41 on platelet derived MPs and the ''non detection '' of CD5 on CLL B-cell derived MPs by immunogold labeling (S3 Fig). ",
"section_name": "Immunophenotypic characterization of blood cells and blood cell-derived MPs by direct flow cytometry",
"section_num": null
},
{
"section_content": "To confirm the results obtained by direct flow cytometry, we studied MPs derived from three cell types (platelets, BM-MSC, and B-cells) with an optimized latex bead technique.Three different labels were applied to these MPs (Fig 4).Using this method, as previously described, we confirmed the expression of CD41, a platelet marker, on platelet-derived MPs (Fig 4I).This antigen was not found on the 2 other populations of MPs (Fig 4C and4F), validating our protocol.Regarding the direct flow cytometry, positive signals were observed for some markers (CD19-PE on B cell-derived MPs, CD90 on MSC-derived MPs).However, when the different MPs were cross-labeled for these markers (CD19 on MSC-derived MPs and CD90 on B-cell-derived MPs), a positive signal was also observed, indicating that the signals were false positives. ",
"section_name": "Immunophenotypic characterization by latex bead flow cytometry",
"section_num": null
},
{
"section_content": "Microvesicles play a crucial role in cellular interactions and have been considered a novel mechanism of cross-talk between normal and malignant cells [4].Even if the phenotypic characterization of extracellular vesicles is well defined in the literature, there are some discrepancies concerning antigen detection.Besides, Oksvold et al. described that there is an important difference in subpopulations of exosomes derived from the same B-cell lymphoma cell line expressing their own panel of proteins [56].In the present study, we attempted to clarify the differences found in the literature about the phenotype of several blood cell-derived MPs and we proposed a new negative control to avoid false positive detection.The expression of different antigens (CD3, 5, 8, 11c, 14, 16, 19, 20, 27, 41, 56, and 61) was investigated on B-lymphocytes, T-lymphocytes, monocytes, NK-cells, platelets, CLL B-cells and their derived MPs by two different flow cytometry techniques. We used Megamix PLUS-beads to calibrate the Navios cytometer, several authors discussed about the impact of the refractive index difference between the beads and the MPs (n = 1.6/ 1.4).This point was clarified and well-argued by Robert et al, they performed differential filtrations using specific filters and proved that platelet MPs enumerated in the MP gate (0.3-1μm) were not affected by 1μm filtration, but completely depleted by 0.2μm filtration.They concluded that difference of index doesn't affect extensively the flow cytometry forward scatter (FCM FS) results [52;60].However, it should be noted that this subject is still under debate: some authors highlighted that flow cytometry with polystyrene/latex bead calibration can lead to an underestimation of the vesicle sizes.It should be interesting to use silica beads that have refractive index much closer than biological MVs.Even if the NAVIOS cytometer is currently one of the best cytometers [52] current flow cytometers have some limitations and are only capable to measure the ''top of the iceberg\".The vesicle sizes must be considered with caution [61][62][63][64][65] To keep MPs as close to their native state as possible, we did not use any activator to increase MP production, in contrast to previous work that used products such as calcium ionophore 23187, LPS and collagen [66].Connor et al and Sims et al demonstrated that the phenotype of platelet-derived MPs can vary depending on the nature of activator used to induce their formation; notably collagen, thrombin, ADP or calcium ionophore [21;67].Similar observations were reported by Jimenez et al for endothelial-cell-derived MPs [68] and by de Jong et al for exosomes [45].In our study, MPs were isolated by ultracentrifugation, while filtration steps were avoided as recommended by Macey et al [36]. We considered MPs as all events within the MP gate and positive for annexin-V labeling.Although this marker is widely used in the MP field [35;52;69], the subject remains under debate.Indeed, the use of phosphatidylserine (PS) staining is not always applied to define MP populations [21;22;24;25;28].Therefore, we analyzed both populations of MPs (annexin-V positive and negative). Our study confirmed that CD41/CD61 expression on platelet-derived MPs can be detected by direct and latex bead flow cytometry as it was largely described in the literature [5;6].Surprisingly, we obtained a weak positive signal for specific markers such as CD3, CD19, and We calculated the mean of the MFIR ± SEM (Mean fluorescence intensity ratio ± standard error of the mean) for all used antibodies (n = 5).The expression of all antibodies was evaluated on 5 cell type-derived MPs, both annexin-V + and-populations and also on the original cells.These numbers demonstrated that antigens are clearly expressed on the cells but not necessary on the MPs. doi:10.1371/journal.pone.0127209.t001 CD56 for T-lymphocytes, B-lymphocytes and NK-cell-derived MPs, respectively.This labeling was shown to be a false positive using the same markers on MPs derived from different cell types (Fig 3).Moreover no other specific antigens were detected on MPs while these antigens were significantly found on the source cells.This was also confirmed with a different and more sensitive technique (immunogold labeling).We were able to detect CD41 on platelet derived A recent report suggested that there is a major decrease in antigen expression between cells and EVs [71].Moreover, it has already been shown that MPs are derived from lipid rafts and we can thus hypothesize that these parts of the membrane can be characterized by differential partitioning of antigens [72][73][74][75].Miguet et al observed by a proteomic study that while some antigens are detected on MPs, CD3, 5, and 8 for T-cells and CD19 for B-cells are not found on MPs despite being highly expressed on the original cells [76].Our results conflict with those of authors who report detection by direct flow cytometry of CD105, CD90, and CXCR4 on MPs from mesenchymal stem cells [77] and CD3, CD14 and CD19 on MPs from the plasma of polymyositis/dermatomyositis patients [78].In addition, Ghosh et al and Macey et al detected annexin-V-CD19+ [35] and CD15+ MPs [36], respectively.These discrepancies with our study could be explained by several factors: speed of centrifugation, choice of specific negative controls and antibodies, MP origin and the presence of specific activators in culture.Indeed, these authors analyzed MPs from cell culture or directly from plasma and they either applied centrifugation forces of 16,000 x g to 19,800 x g for 10 min to 1 h or they did not use centrifugation at all.More importantly, all of these groups used different negative controls.Baka et al used unstained samples and Kim et al, Ghosh et al, Macey et al used isotype controls.Trummer and colleagues demonstrated that using isotype negative controls can induce errors: they concluded that using flow cytometry to discriminate between positive and negative populations of MPs labeled with several antibodies could be an obstacle for their characterization [79].All of these differences could explain the variation in results between several groups.The study of MPs is complicated by their limited surface area and relatively small number of proteins available for antibody binding.Thus, the latex bead technique has been used by several groups to facilitate the characterization of MPs by flow cytometry.Different protocols have been published [37;54;55], but we adapted the protocols of Wu et al and Mokarizadeh et al with minor modifications [39;40].Since no significant difference was observed by using blocking reagent, we removed this step, as previously described by Oksvold et al [56].Three different negative controls were used: beads alone without any staining, beads with MPs and isotype control and beads with MPs and a negative marker (one not present on the original cells).Isotype controls were removed from our study because they resulted in some false positive results (data not shown).Szczepanski et al, Wu et al and Mokarizadeh et al presented positive results from the first protocol, notably for CD9, CD33, CD63, CD73, and CD90 [39;40;54].However, in our study, we demonstrated that this protocol is associated with non-specific labeling due to antibody affinity for latex beads.Indeed incubating beads with antibody in the absence of MPs resulted in an increase of background noise (data not shown).Thereafter, we designed our own protocol, by directly incubating the beads with previously labeled MPs.In this way, we avoided an increase in background signals due to non-specific binding of antibodies to beads.Using this protocol we were able to detect specific markers on platelet-derived MPs.Together, our results indicated that several previously published data sets using the latex bead technique include false positive results due to the use of inappropriate protocols or negative controls. The goal of this study was to emphasize on the inconsistencies in literature concerning the characterization of MPs by flow cytometry and the importance of the negative control choice.We confirmed the data of Trummer and colleagues concerning the failure of classical negative control (isotype) in the MP study.Since, some antigens could be expressed by different cell types (i.e.CD5 could be found on T or B cells), several negative controls should be tested for a specific MV staining. In conclusion, the characterization of MPs is still a challenging field requiring lot of precautions in the interpretation of the fluorescence signal.We demonstrated that isotype controls and unstained samples are not suitable for MP characterization.Therefore, we proposed using other negative markers (marker not found on the original cell) by cross-staining MPs to demonstrate the true positive labeling.We want to underline the fact that this method is applicable on cell culture supernatants specifically but not on body fluids (like urine or plasma), which are more complex samples.By this manner, non-specific antibody labeling could be subtracted out, and reliable results could be obtained. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "This research was supported by a Télévie and a FRSM (Fonds de la Recherche Scientifique Médicale) grant, both affiliated to the \"Fonds National de la Recherche Scientifique -FNRS\" (F.R.S-F.N.R. S.). ",
"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
}
] |
10.1186/s12967-021-03136-2
|
Ibrutinib protects T cells in patients with CLL from proliferation-induced senescence
|
<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>The development of Bruton’s tyrosine kinase inhibitors (BTKi) for the treatment of chronic lymphocytic leukaemia (CLL) has provided a highly effective and relatively non-toxic alternative to conventional chemotherapy. Some studies have shown that BTKi can also lead to improvements in T cell immunity in patients despite in vitro analyses suggesting an immunosuppressive effect of BTKi on T cell function.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>In this study, we examined both the in vitro effect and long-term in vivo effect of two clinically available BTKi, ibrutinib and zanubrutinib. Additional in vitro assessments were undertaken for a third BTKi, acalabrutinib. Immune subset phenotyping, cytokine secretion, T cell degranulation and proliferation assays were performed on peripheral blood mononuclear cells isolated from untreated CLL patients, and CLL patients on long-term (> 12 months) BTKi treatment.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Similar to prior studies we observed that long-term BTKi treatment normalises lymphocyte subset frequency and reduces PD-1 expression on T cells. We also observed that T cells from patients taken prior to BTKi therapy showed an abnormal hyper-proliferation pattern typical of senescent T cells, which was normalised by long-term BTKi treatment. Furthermore, BTKi therapy resulted in reduced expression of the T cell exhaustion markers PD-1, TIM3 and LAG3 in late generations of T cells undergoing proliferation.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>Collectively, these findings indicate that there are critical differences between the in vitro effects of BTKi on T cell function and the effects derived from long-term BTKi exposure in vivo. Overall long-term exposure to BTKi, and particularly ibrutinib, resulted in improved T cell fitness in part due to suppressing the abnormal hyper-proliferation of CLL T cells and the associated development of T cell senescence.</jats:p></jats:sec>
|
[
{
"section_content": "Chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia in the western world and is associated with significant perturbations of cellular and humoral immunity including an excess of terminally differentiated T cells and elevated expression of exhaustion markers including Program cell Death molecule-1 (PD-1) and Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) (Reviewed in [1]).Compounding these CLL-specific immune defects are the additional immunosuppressive effects of prior lympholytic conventional chemotherapy, homeostatic proliferation of dysfunctional post-chemotherapy T cell clones [2] and the underlying effects of aging-related T cell senescence (Reviewed in [3]).More recently, Bruton's tyrosine kinase (BTK) inhibitors, particularly the first generation BTK inhibitor (BTKi) ibrutinib, have become a mainstay CLL therapy.Initially indicated for the treatment of relapsed CLL, [4] more recently BTKi has been incorporated as part of frontline therapy [5] and thereby represent an opportunity for chemotherapy-free treatment of CLL, potentially reducing the impact of lymphodepleting chemotherapy and promoting long-term preservation of immunity.In addition, ibrutinib also inhibits other Tec family kinases, including IL-2-inducible T cell kinase (ITK) in T cells, leading to enhanced T H 1 immune responses [6].The second generation more selective BTKi, including zanubrutinib and acalabrutinib, have similar clinical efficacy in control of CLL [7][8][9] but with limited ITK inhibition and therefore potentially a decreased impact on T cell function. Although there is substantial evidence of the negative impact of ibrutinib-induced suppression of NK cell function [10], including inhibition of ADCC [11], the direct impact of ibrutinib or other BTKi on the in vitro function of T cells has been less well described.However, ibrutinib is known to inhibit the activation, degranulation and proliferation of healthy donor T cells [12].In a mouse model of CLL ibrutinib treatment resulted in significantly lower numbers of CD8 + effector T-cells, with lower expression of activation markers, as well as impaired proliferation and effector function.However, co-administration of ibrutinib was associated with increased efficacy of checkpoint blockade therapy and improved CD8 + T-cell effector function and control of CLL suggesting that modulation of T cell function by ibrutinib may lead to enhanced T cell function in vivo [13].Clinically, early observations in ibrutinib-treated patients suggested an immunosuppressive effect with increased opportunistic infections in heavily pre-treated patients with CLL who subsequently were treated with ibrutinib [14][15][16][17].These findings have been linked to the impairment of macrophage function by ibrutinib [18,19] in addition to cumulative impairment of cellular immunodeficiency induced by prior therapies and/or persistent immune dysfunction exerted by the effects of the CLL microenvironment.Importantly, there appear to be few opportunistic infections recorded in previously untreated patients with CLL who receive ibrutinib as their first therapy [20].Indeed, frontline ibrutinib therapy has been associated with a broadening of T cell repertoire and a lower rate of opportunistic infections [21,22].Furthermore, others have shown that ibrutinib therapy, but not therapy with the more selective second generation BTKi acalabrutinib, over the course of 6 cycles of treatment is associated with improved CD4 + and CD8 + T cell numbers, possibly by preventing ITK-dependent activation-induced T cell death [23].This study also showed a differential BTKi effect of ibrutinib-induced reduction of expression of the T cell suppression surface markers PD-1 and CTLA-4 and overall suggested that ibrutinib exposure improves the number and functional status of T cell populations.More recently it has been reported that long-term treatment with ibrutinib significantly restored T-cell proliferative ability, degranulation, and cytokine secretion in peripheral blood samples from patients with CLL [24].We have also demonstrated that long-term exposure to ibrutinib in vivo may return the T cell function of patients with advanced mantle cell lymphoma (MCL) to that of healthy donors [25].Overall, this improved T cell fitness following ibrutinib therapy may in turn be able to be exploited to improve the generation and efficacy of subsequent adoptive CAR-T cell therapy in CLL [26] or other B cell malignancies including MCL [27]. In this study we wished to explore the impact of first and second generation clinically available BTKi (ibrutinib, acalabutinib and zanubrutinib) on T cell function in vitro and assess whether those effects translated to changes in post-proliferation T cell exhaustion following long-term exposure to BTKi in patients with CLL. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Blood samples were collected from CLL patients whose disease was refractory to previous treatment and were about to commence either single-agent ibrutinib or zanubrutinib therapy.CLL patients were treated with ibrutinib under compassionate access at the Peter MacCallum Cancer Centre, Melbourne, Australia or zanubrutinib under a Phase I clinical trial (NCT02343120 [9]).Baseline samples were collected prior to treatment with the specified BTKi and long-term treatment samples were collected after 12 months on zanubrutinib therapy (n = 8), and between 12-24 months on ibrutinib therapy (n = 7) (Additional file 2: Table S1).Age-matched Healthy Donor (n = 7) samples were used in ex vivo analysis comparing baseline with long-term treatment samples from CLL patients.In vitro analysis examining the effects of shortterm BTKi treatment on T cell function used samples from a separate cohort of Healthy Donor (n = 6), and treatment-naive CLL patients (n = 11). ",
"section_name": "Patient cohort",
"section_num": null
},
{
"section_content": "Peripheral blood samples were collected with informed consent from CLL patients at the Peter MacCallum Cancer Centre and The Royal Melbourne Hospital (Melbourne, Australia) under ethics approval of the respective institution's human research ethics committee and in accordance with the Declaration of Helsinki.Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Paque plus (GE Healthcare, Chicago, IL) density gradient separation and cryopreserved until required.Peripheral blood samples from age matched Healthy Donor were obtained from the Australian Red Cross Blood Service with ethics approval from the Melbourne Health Human Research Ethics Committee. ",
"section_name": "Patient sample processing",
"section_num": null
},
{
"section_content": "In vitro experiments were performed in this study using HD or treatment naive CLL patient PBMC treated with vehicle or BTKi during the experiment, whereas ex vivo experiments were performed using PBMC from patients collected at baseline or after long-term treatment on BTKi to examine the effect of targeted therapies on immune cell profiles and function.For in vitro analyses, PBMC were thawed and cultured at 1.0 × 10 6 / mL in T cell media (TCM) (Gibco RPMI media 1640, 10% v/v heat inactivated foetal calf serum (FCS), 1% v/v 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 × Gibco GlutaMAX, 1 × Gibco MEM nonessential amino acids, 1 mM Gibco sodium pyruvate, 100 U/mL Penicillin/Streptomycin (all from Thermo Fisher Scientific, Watham, MA) and 50 mM 2-Mercaptoethanol (2ME) (Sigma Aldrich, St Louis, MO)) supplemented with 1 µM ibrutinib (Selleck Chemicals), 1 µM zanubrutinib (BGB-3111, Obtained under a Materials Transfer Agreement with BeiGene Co. Ltd.) or 1 µM acalabrutinib (Selleck Chemicals), or 0.5% DMSO (Sigma Aldrich) vehicle control at 37 °C, 5% CO 2 for 18 h prior to use in experiments.For ex vivo analysis, PBMC were thawed and plated at 1.0 × 10 6 /mL in TCM, and used immediately.For all flow cytometry based assays, samples were acquired on a BD LSRFortessa Flow Cytometer (BD Biosciences).Analysis was performed using FlowJo software v10.7.1 (BD Biosciences). ",
"section_name": "Cell culture",
"section_num": null
},
{
"section_content": "For both the in vitro and ex vivo studies PBMC were activated for four hours with CD2/CD3/CD28 T cell activation beads (Miltenyi Biotec, Cologne, Germany) in the presence of GolgiStop and GolgiPlug protein transport inhibitors (BD Bioscience, Franklin Lakes, NJ), and antihuman CD107a APC conjugated antibody (clone H4A3, BD Bioscience).Cell surface marker staining was performed for the in vitro studies using LIVE/DEAD Aqua fixable dead cell stain (Thermo Fisher Scientific), CD4 APC Cy7 (clone RPA-T4, BD Bioscience), CD3 BV785 (clone OKT3, BioLegend, San Diego, CA) CD8α BV650 (clone RPA-T8, BioLegend), and IFNγ PE (clone B27, BD Biosciences).Cell surface marker staining was performed for the ex vivo study using LIVE/DEAD Aqua fixable dead cell stain, CD4 BUV395 (clone SK3, BD Biosciences), CD3 BUV496 (clone UCHT1, BD Biosciences) and CD8α BUV805 (clone SK1, BD Biosciences).Surface and intracellular cytokine staining was performed using the Cytofix/Cytoperm kit (BD Biosciences) according to manufacturer's instructions. ",
"section_name": "Degranulation assay",
"section_num": null
},
{
"section_content": "PBMC were stained with 5 μM CellTrace violet (CTV, Thermo Fisher Scientific) according to manufacturer's instructions and stimulated with CD2/CD3/CD28 T cell activation beads.For in vitro studies, on days 3 and 5 half of the media was removed from the culture, and 20 IU/mL rhIL-2, and 1 µM BTKi or 0.5% DMSO added in TCM.Cells were harvested on days 3, 5, and 7 for T cell proliferation and cytokine production analysis.For ex vivo studies, on day 3 half of the media was removed from the culture and 20 IU/mL rhIL-2 in TCM was added.Cells were harvested on day 5 for T cell proliferation analysis.Cell surface marker staining was performed using LIVE/DEAD Aqua fixable dead cell stain, CD19 PECy7 (clone SJ25-C1, BD Biosciences), CD4 BUV395 (clone SK3, BD Biosciences), CD3 BUV496 (clone UCHT1, BD Biosciences), CD8 BUV805 (clone SK1, BD Biosciences), CD27 FITC (clone M-T271, BD Biosciences), CD45RA PerCPCy5.5 (clone HI100, Thermo Fisher Scientific), LAG3 AF647 (clone T47-530, BD Bioscience), PD-1 BV786 (clone EH12.1, BD Bioscience), and TIM3 PE (clone 7D3, BD Bioscience).The flow cytometry panel described above was also used for phenotypic analysis of freshly thawed PBMC (Additional file 1: Figure S1). Proliferation of CD4 + and CD8 + T cells was measured using FlowJo (BD Bioscience) cell proliferation analysis to identify up to 9 generations of dividing cells based on CTV intensity.Generation 0 represented undivided cells, and generation 9 represented cells that had undergone at least 8 divisions.The percentage of cells in generations 0, 1, and 2 was added to give a combined total percentage of cells in generations 0-2, representing undivided and newly dividing cells (Additional file 1: Figure S2).The median fluorescence intensity (MFI) of PD-1, TIM3 and LAG3 expression was calculated for individual generations of dividing CD4 + and CD8 + T cells. ",
"section_name": "T cell proliferation assay and phenotypic analysis",
"section_num": null
},
{
"section_content": "PBMC were stimulated with CD2/CD3/CD28 T cell activation beads for 24 h to 7 days, and tissue culture media collected.The BD Cytometric Bead Array (CBA) Human Th1/Th2/Th17 cytokine kit (BD Biosciences) was used to quantify the concentration of IL-2, IL-4, IL-6, IL-10, IL17A, IFNγ and TNFα, according to the manufacturer's instructions.The sensitivity of the assay was 5000 pg/mL and therefore any readings that fell above this level were censored to 5000 pg/mL.CBA analysis was performed using FCAP Array v3.0 software (BD Biosciences). ",
"section_name": "Cytokine bead array",
"section_num": null
},
{
"section_content": "Prism V9.0 (GraphPad) was used to perform statistical analysis in this study.For the in vitro experiments, statistical analysis was performed using Mixed effects analysis with Tukey-Kramer test for multiple comparisons for the degranulation and ICS experiments; RM oneway ANOVA with Fisher's LSD for cytokine production; Friedman test, Dunn's multiple comparisons for exhaustion marker expression; and Two-way ANOVA with Fisher's LSD multiple comparisons for CTV T cell generation analysis.For the ex vivo experiments, statistical analysis was performed using Mann-Whitney unpaired T tests between the Healthy Donor and CLL patient samples, and Wilcoxon matched pairs T test between the baseline and long-term treatment CLL patient samples.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "In order to examine the impact of BTKi on T cell function in CLL, peripheral blood samples taken at baseline and following long-term treatment (12-24 months) were analysed.CLL patients treated with BTKi showed a significant reduction in the frequency of circulating CD19 + B cells, consistent with the clinical responses observed following long-term ibrutinib or zanubrutinib treatment (Fig. 1A), however most patients still had detectable tumour cells.As described by others [23,24], a commensurate increase in the percentage of CD3 + T cells following long-term BTKi therapy was observed (Fig. 1B).This was not associated with a change in CD4:CD8 T ratio (Fig. 1C), nor an alteration in the frequency of memory subsets in CD8 + T cells (Additional file 1: Figure S3A-D).In contrast, CLL patients had low frequencies of CD4 + naïve T cells compared to healthy donors, which significantly increased after long-term zanubrutinib treatment (Additional file 1: Figure S4A-D).Exhaustion marker profiling confirmed that T cells isolated from CLL patients prior to BTKi treatment had increased expression of PD-1 on CD4 + and CD8 + T cells compared with healthy donors (Fig. 1D,E) which significantly decreased in CD4 + T cells after long-term zanubrutinib treatment. Ex vivo T cell assays indicated that there were no effects of long-term BTKi treatment on the ability of CD4 + or CD8 + T cells to degranulate (Fig. 2A,B).This suggested that functional analysis using degranulation may provide an inaccurate measure of drug-induced phenotypic changes in T cell behaviour.We therefore investigated the impact of drug therapy on the ability of T cells to proliferate.Compared to healthy donor T cells, the baseline samples from CLL patients showed increased proliferation resulting in a decreased percentage of cells in early generations (generations 0-2) for CD4 + T cells which was corrected to healthy donor levels with ibrutinib treatment (Fig. 2C,D).A similar trend was seen in CD4 + T cells in patients treated with zanubrutinib and in CD8 + T cells treated with either BTKi however this did not reach statistical significance (Fig. 2D,E).Collectively, this data suggests that long-term BTKi exposure results in a reduction in hyperproliferative T cell responses, and may contribute to normalisation of T cell phenotype independent of off-target ITK inhibition. ",
"section_name": "T cells improve in number and function after long-term BTKi exposure",
"section_num": null
},
{
"section_content": "To explore the mechanisms leading to the observed recovery of T cell numbers, we sought to determine if the various BTKi altered CD8 + T cell effector functions in vitro.It has been previously demonstrated that NK cell degranulation is suppressed by ibrutinib, but not by the more selective BTKi [10,11,28].Therefore, we investigated the impact of short-term in vitro ibrutinib, zanubrutinib or acalabrutinib treatment on T cell function, in treatment naïve CLL patient PBMC.CD8 + T cell degranulation was significantly inhibited by ibrutinib, compared to the DMSO vehicle control (Fig. 3A).Intracellular IFNγ accumulation was similarly reduced in ibrutinib treated cultures in both CD8 + and CD4 + T cells (Fig. 3B and Additional file 1: Figure S5).Neither zanubrutinib nor acalabrutinib significantly altered CD8 + T cell degranulation or IFNγ expression.Interestingly, ibrutinib treated CLL PBMC showed a similar level of CD8 + T cell degranulation as healthy donors.However, ibrutinib also inhibited CD8 + T cell degranulation and IFNγ expression in healthy donor cultures (Additional file 1: Figure S6) suggesting that the inhibition of T cell degranulation responses by ibrutinib is due to the inhibition of intrinsic CD8 T cell responses, potentially attributable to ITK inhibition by ibrutinib decreasing TCR signalling, rather than alteration to the extrinsic environment (via BTK inhibition in other PBMC subsets). To further assess the impact of BTKi on short-term T cell functional responses, a 24-h cytokine release assay demonstrated that in vitro treatment with ibrutinib significantly reduced IL-2 and IFNγ secretion by CLL patient PBMC (Fig. 3C).A similar pattern was observed in healthy donor PBMC, in which ibrutinib, but not the more selective BTK inhibitors, resulted in significantly decreased levels of IL-2, IFNγ and TNFα (Additional file 1: Figure S6).These findings further support the conclusion that inhibition of ITK by ibrutinib significantly alters T cell function, while more specific BTKi do not significantly impact intrinsic T cell responses to TCR mediated stimuli. Next, we examined the effect of BTKi on T cell proliferation.Over 7 days, only ibrutinib, but neither zanubrutinib nor acalabrutinib, significantly reduced CD4 + and CD8 + T cell proliferation (Fig. 3D,E).Strikingly, short-term treatment of ibrutinib in CLL patient PBMC reduced abnormally high T cell proliferation rates to that of healthy donor levels.These findings suggest that while ibrutinib treatment diminishes the magnitude of TCR mediated T cell responses, long-term in vivo treatment with BTKi may restore T cell fitness and ability to respond to subsequent T cell activation. ",
"section_name": "Short-term ibrutinib treatment impairs T cell function in vitro",
"section_num": null
},
{
"section_content": "Patients treated with both ibrutinib and more selective BTKi have significantly reduced expression of co-inhibitory ligands on patient T cells [23,29].We therefore sought to understand if this effect was recapitulated after in vitro BTKi treatment of proliferating T cells.Ibrutinib treated CD8 + T cells had significantly decreased expression of PD-1, TIM3 and LAG-3 after 3-and 5-days and TIM3 and LAG-3 after 7-days stimulation, while zanubrutinib and acalabrutinib had no significant effect on co-inhibitory ligand expression (Fig. 4).Notably, ibrutinib treated CD8 + T cells displayed normalised exhaustion marker expression, similar to the levels observed in age-matched healthy donor T cells.This is the first study to directly compare ibrutinib, acalabrutinib and zanubrutinib treatment on PD-1, TIM3 and LAG3 expression on proliferating T cells from patients with CLL, and highlights the differential impacts of different BTKi on T cell function. Considering that short-term in vitro ibrutinib treatment inhibited T cell proliferation (Fig. 3E), we next explored if changes in the proportion of proliferating cells was associated with decreased exhaustion marker expression.Utilising proliferation gating we compared the expression of exhaustion markers on CD8 + and CD4 + T cells which had undergone multiple divisions after 5 days stimulation.CD8 + T cells treated with ibrutinib had significantly lower expression of TIM3 and LAG3 than control treated counterparts, approaching the levels of expression observed in healthy donor CD8 + T cells (Fig. 5A).The effect of ibrutinib treatment on the expression of PD-1, TIM3, and LAG3 on CD4 + T cells was less pronounced, however early generations of CD4 + T cells had significantly decreased PD-1 expression while later generations had a more normalised expression of TIM3 (Fig. 5B).Notably, zanubrutinib and acalabrutinib did not have a significant effect on PD-1 or LAG3 expression in either CD8 + or CD4 + T cells, however both decreased the expression of TIM3, albeit to a lesser degree than ibrutinib. To investigate if the decreased expression of PD-1, TIM3 and LAG3 on CD8 + T cells induced by ibrutinib was due to pre-existing CLL patient T cell exhaustion, the effects of BTKi on the proliferation of healthy donor cells was analysed.Healthy donor CD8 + T cells treated with ibrutinib exhibited decreased expression of PD-1 and LAG3 compared to vehicle control.However, this effect was restricted to early generations, and in later generations ibrutinib treated CD8 + T cells had increased expression of PD-1 and, along with zanubrutinib treated cells, TIM3 (Fig. 5C,D).Together this data suggests that while ibrutinib decreases the expression of exhaustion markers in already exhausted CLL patient T cells, the effect in healthy donor T ",
"section_name": "Short-term ibrutinib treatment impairs T cell proliferation and exhaustion in vitro",
"section_num": null
},
{
"section_content": "To confirm that ibrutinib exposure resulted in T cell populations that were less likely to undergo senescence following proliferation, we examined exhaustion marker expression on proliferating T cells collected from CLL patients prior to, and after long-term BTKi exposure.Exhaustion marker analysis on T cells after 5 days of stimulation indicated that CD4 + and CD8 + T cells from CLL patient baseline samples had elevated expression of PD-1 compared to healthy donor T cells (Additional file 1: Figure S7).After long-term treatment with zanubrutinib, expression of PD-1 significantly decreased on both CD4 + and CD8 + T cells, towards healthy donor levels.Generational gating was used on individual CD4 + and CD8 + T cell populations to dissect out the change in exhaustion marker expression with cell division.The expression of PD-1 and LAG3 was highly elevated in the multiply divided T cells from baseline samples collected from CLL patients, compared to healthy donor T cells (Fig. 6), indicative of the formation of an exhausted pool of T cells.Long-term treatment of CLL patients with either ibrutinib or zanubrutinib resulted in a significant decrease of PD-1 and LAG3 expression in dividing T cells, and an increase of TIM3 expression, thus resulting in a T cell population with a less exhausted phenotype despite persistent circulating tumour burden and ongoing antigen stimulation (Fig. 1A). ",
"section_name": "Long-term BTKi exposure reduces T cell senescence",
"section_num": null
},
{
"section_content": "The development of BTKi drug therapy has substantially improved the efficacy and reduced the toxicity of CLL treatment.The use of BTKi appears to result in a clinical improvement in immunity with less observed opportunistic infections compared to historical cohorts treated with conventional chemotherapy.Indeed, others have recently shown that these clinical observations are supported by a measured increase in CD4 + and CD8 + T cell numbers and a lowered expression of exhaustion markers, particularly PD-1 in patients treated with ibrutinib, but not the more selective BTKi drug acalabrutinib [23].In contrast, total CD4 + and CD8 + T cells have been reported to be unchanged in patients treated with zanubrutinib but these cells exhibit reduced PD-1 expression [30].Understanding the mechanisms underpinning this apparent improvement in T cell fitness is important, not only because of its wider implications for supportive care of patients with CLL, but also for the optimal timing of immunotherapy in CLL and other low grade B cell malignancies.Whilst the impact of cellular and T cell-engaging immunotherapy in these diseases has initially been disappointing, their role is being re-explored in the context of earlier application or in combination with initial non-chemotherapeutic debulking strategies, including in combination with BTKi.Both approaches may result in enhanced efficacy of immunotherapy through exploiting a less exhausted and fitter effector T cell population, which has been recently identified as a critical determinant of immunotherapy responses [31]. Importantly, the exploration of the impact of BTKi on T cell function may be misleading if interpretation is solely reliant on short-term in vitro analyses.As we and others [23] have demonstrated, in vitro ibrutinib treatment results in suppression of key T cell functions from both CLL and healthy donors including TCR-mediated proliferation, cytokine production and degranulation (Fig. 3 and Additional file 1: Figures S5,S6), the last of which is a reliable surrogate maker for T cell cytotoxic capacity [32].However, given the baseline abnormal hyperproliferative response seen in CLL T cells, treatment with ibrutinib, but not other BTKi, actually results in a reduction in proliferation to that seen in healthy donor cells (Fig. 3D).This may explain the fact that despite these apparently suppressive effects on T cell function, we and others have consistently observed an increase in CD3 + T cell numbers following long-term treatment with ibrutinib but to a much lesser extent with acalabrutinib [23] or zanubrutinib (Fig. 1).Whilst the frequency of T cells in the blood was increased after long-term BTKi therapy as a result of the reduction of CLL burden in the patients we analysed, none had complete clearance of circulating tumour cells despite many months of treatment.Given this, we feel that our findings represent a direct effect of BTKi therapy onthe function and phenotype of T cells rather than as a function of tumor debulking alone. We wished to explore further these apparently contradictory findings of short-term in vitro suppression of T cell function and their long-term recovery following exposure to ibrutinib.We have been able to assess the impact of ibrutinib, acalabrutinib and zanubrutinib on the T cell phenotype and function in vitro and additionally examine the effects of ibrutinib and zanubrutinib on two cohorts of patients treated at our centre. Our findings indicate that ibrutinib-induced suppression of T cell proliferation can prevent T cell exhaustion, and contribute to an increased CD3 + T cell pool.In an analysis of the expression of the exhaustion markers PD-1, TIM3 and LAG3, we identified that the expression of all three decreased on CD4 + and CD8 + T cells from untreated patients with CLL after three days of culture in the presence of ibrutinib, but not acalabrutinib or zanubrutinib.Others have suggested that ibrutinib-induced suppression of PD-1 may indicate protection of T cells from activation-induced cell death [23].In our analysis, a longitudinal time course of in vitro culture indicated that PD-1 suppression by ibrutinib may be transient, whereas suppression of LAG3 and TIM3 is more durable (Fig. 4).As indicated previously these effects appear to be ibrutinib-specific.The development of an exhausted T cell phenotype is most likely to occur in the setting of chronic inflammation-induced T cell proliferation including that induced by aging, chronic infection and malignancy, and may underpin the T cell immunosuppression associated with these conditions [3].We reasoned that suppression of abnormal T cell proliferation should also be associated with avoidance of exhaustion potentially independent of tumour control.Indeed, in an analysis of proliferation, ibrutinib reduced the proportion of late generation CD8 + T cells following CD3/CD28 bead stimulation, which showed a significantly lower expression of the exhaustion markers LAG3 and TIM3 (Figs. 4 and5).A lesser effect was seen with acalabrutinib and zanubrutinib and with all three drugs in CD4 + T cells (Fig. 4).This same pattern was seen in the T cells examined from patients treated with ibrutinib, and to a lesser extent with zanubrutinib for 12-24 months (Fig. 6) indicating that BTKi therapy protected the T cell population present at the time of treatment initiation from the development of an exhausted phenotype.This is further highlighted by the observation of retention of normal degranulation following long-term ibrutinib treatment (Fig. 2). Collectively our data show that ibrutinib can induce reduction in critical T cell functions when assessed in short-term in vitro cultures.These findings belie the observations of an improved T cell repertoire and improved clinical immune function after long-term therapy with ibrutinib.This improvement stems from the ability of ibrutinib to protect T cells from the development of an exhausted phenotype that follows proliferation and maintenance of an enriched naïve T cell population.These effects seem to require the offtarget suppression of ITK by ibrutinib as the effects are not observed following acalabrutinib or zanubrutinib therapy (Fig. 3).In this sense the less specific effects of ibrutinib are important for the observed T cell effects and may underpin the recent findings that ibrutinib improves the production of CAR-T cellsin patients with CLL previously treated with ibrutinib [26] and that ibrutinib supplementation during CAR-T production results in a greater cell yield with a more naïve-like phenotype and decreased expression of exhaustion markers [33].Indeed, based on these findings a number of trials have been initiated utilising BTKi in combination with CAR-T administration in MCL (NCT04234061) and CLL (NCT03960840).Faced with a choice of BTKi, and with an opportunity to avoid the off-target side effects associated with ibrutinib, the use of more specific BTKi therapy may inadvertently miss out on the opportunities for improvement in T cell fitness.In this context, ibrutinib may be increasingly considered an immunomodulatory drug that can be applied outside of B cell malignancies.Indeed, the immunomodulatory effect of ibrutinib on 'off target' immune cells is being investigated for the treatment of metabolic inflammatory disorders such as Type 2 Diabetes [34]. Overall, our findings indicate that a programmatic approach to CLL and other B cell malignancies should be considered, whereby ibrutinib therapy for both disease control and improved T cell fitness is followed immediately by immunotherapy in the form of either CAR-T cell therapy or BiTE therapy.This two-pronged approach will permit clearance of residual tumour cells prior to relapse and exploit the more functionally normal, and therefore more fit, T cell repertoire in order to achieve deeper and more durable responses and potentially a cure in these previously incurable malignancies. Additional file 1: Figure S1.Flow cytometry gating strategy.PBMC from HD or CLL patients were gated on single cells, lymphocytes, live cells, and CD19 + B or CD3 + T cells.The memory phenotype of CD8 + or CD4 + T cells was measured using Naïve (N, CD27 + CD45RA +), Central memory (CM, CD27 + CD45RA-), effector memory (EM, CD27-CD45RA-), and terminally differentiated effector memory (TEMRA, CD27-CD45RA +) subsets.CD4 + and CD8 + T cells were also tested for the expression of PD-1, TIM3 and LAG3.Flow gating in the bottom row shows representative CD8 + T cell subsets.Figure S2.Generation 0-2 cell proliferation analysis.Proliferation of CD4 + and CD8 + T cells was measured using FlowJo cell proliferation analysis to identify 9 generations of dividing cells.Generation 0 represents undivided cells, and generation 9 represents cells that have undergone at least 8 divisions.The percentage of cells in generations 0, 1, and 2 was added to give a combined total % of cells in generations 0-2, representing undivided or early generation T cells.Figure S3.Stable memory phenotype CD8 + T cell subset frequency in CLL patients on long-term BTKi therapy.PBMC from CLL patients collected prior to treatment (baseline), or after long-term treatment of ibrutinib (n = 7) or zanubrutinib (n = 8) were compared to Healthy Donor (HD, n = 7).Naïve (N, CD27 + CD45RA +), Central memory (CM, CD27 + CD45RA-), effector memory (EM, CD27-CD45RA-), and terminally differentiated effector memory (TEMRA, CD27-CD45RA +) cells (A-D) were calculated as a percentage of CD8 + T cells.Figure S4.Increased naive CD4 + T cell subset frequency in CLL patients on long-term BTKi therapy.PBMC from CLL patients collected prior to treatment (baseline), or after long-term treatment of ibrutinib (n = 7) or zanubrutinib (n = 8) were compared to Healthy Donor (HD, n = 7).Naïve (N, CD27 + CD45RA +), Central memory (CM, CD27 + CD45RA-), effector memory (EM, CD27-CD45RA-), and terminally differentiated effector memory (TEMRA, CD27-CD45RA +) cells (A-D) were calculated as a percentage of CD4 + T cells.Figure S5.Ibrutinib inhibits cytokine release in CD4 + T cells from CLL patients in vitro.PBMC from untreated CLL patients (n = 5) were incubated with 1 μM ibrutinib, zanubrutinib, or acalabrutinib, or DMSO vehicle control for 18 h prior to activation with T cell stimulation beads for 4 h.CD4 + T cell intracellular IFNγ production is shown, and statistical analysis was performed using mixed effects analysis with Tukey-Kramer test for multiple comparisons.Figure S6.Ibrutinib inhibits T cell cytotoxicity, cytokine release and proliferation of healthy donor T cells in vitro.PBMC from Healthy Donor (HD, n = 6) were incubated with 1 μM ibrutinib, zanubrutinib, or acalabrutinib, or DMSO vehicle control for 18 h prior to activation with T cell stimulation beads for 4 h.CD8 + T cell degranulation (A) intracellular IFNγ production (B) are shown, and statistical analysis was performed using mixed effects analysis with Tukey-Kramer test for multiple comparisons.IL-2, IFNγ, TNFα and IL-4 levels in the supernatant were measured after 24 h of T cell activation bead stimulation of PBMC from HD (n = 4) in the presence of BTKi (C).The sensitivity of the assay was limited to 5000 pg/ml.Data was analysed using RM one-way ANOVA with Fisher's LSD.Additional file 2: Table S1.CLL patient and healthy donor characteristics. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "We acknowledge the Melbourne Cytometry Platform (Doherty Institute) for access to flow cytometry instruments.We would like to thank Jenny Collins for assistance with sourcing clinical data. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This study received part funding from the CLL Global Research Foundation and Royal Melbourne Hospital Foundation.KM was supported by a NHRMC Herman Fellowship (APP1090500). ",
"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 and approval from the relevant ethics committees. ",
"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/ s12967-021-03136-2. Study concept/design: CST, DSR, RMK.Acquisition, analysis and interpretation of data: JED, CS, KM.Sourcing of patient samples: CST, KM.Drafting of paper: all authors.Project supervision: DSR, RMK.All authors read and approved the final manuscript. Peripheral blood samples were collected with informed consent from CLL patients at the Peter MacCallum Cancer Centre (project 13/36) and The Royal Melbourne Hospital (Melbourne, Australia-project 2015.128)under ethics approval of the respective institution's human research ethics committee and in accordance with the Declaration of Helsinki.Peripheral blood samples from age matched Healthy Donors were obtained from the Australian Red Cross Blood Service with ethics approval from the Melbourne Health Human Research Ethics Committee (project 2013.288). Competing interests JED, CS, KM: No competing interests.CST: honorarium from Beigene, AbbVie and Janssen.Peter Mac receives research funding from Beigene, AbbVie and Janssen.RMK, DSR: Research funding from BeiGene. • 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 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": "Abbreviations",
"section_num": null
},
{
"section_content": "",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https:// doi.org/ 10. 1186/ s12967-021-03136-2. ",
"section_name": "Supplementary Information",
"section_num": null
},
{
"section_content": "Study concept/design: CST, DSR, RMK.Acquisition, analysis and interpretation of data: JED, CS, KM.Sourcing of patient samples: CST, KM.Drafting of paper: all authors.Project supervision: DSR, RMK.All authors read and approved the final manuscript. ",
"section_name": "Authors' contributions",
"section_num": null
},
{
"section_content": "Peripheral blood samples were collected with informed consent from CLL patients at the Peter MacCallum Cancer Centre (project 13/36) and The Royal Melbourne Hospital (Melbourne, Australia-project 2015.128)under ethics approval of the respective institution's human research ethics committee and in accordance with the Declaration of Helsinki.Peripheral blood samples from age matched Healthy Donors were obtained from the Australian Red Cross Blood Service with ethics approval from the Melbourne Health Human Research Ethics Committee (project 2013.288). ",
"section_name": "Declarations Ethics approval and consent to participate",
"section_num": null
},
{
"section_content": "Competing interests JED, CS, KM: No competing interests.CST: honorarium from Beigene, AbbVie and Janssen.Peter Mac receives research funding from Beigene, AbbVie and Janssen.RMK, DSR: Research funding from BeiGene. • 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": "Consent for publication Not applicable.",
"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
}
] |
10.3389/fonc.2022.909615
|
TP53 Mutations Identified Using NGS Comprise the Overwhelming Majority of TP53 Disruptions in CLL: Results From a Multicentre Study
|
<jats:p>Limited data exists to show the correlation of (tumour protein 53) <jats:italic>TP53</jats:italic> mutation detected by Next generation sequencing (NGS) and the presence/absence of deletions of 17p13 detected by FISH. The study which is the largest series to date includes 2332 CLL patients referred for analysis of del(17p) by FISH and <jats:italic>TP53</jats:italic> mutations by NGS before treatment. Using a 10% variant allele frequency (VAF) threshold, cases were segregated into high burden mutations (≥10%) and low burden mutations (&lt;10%). <jats:italic>TP53</jats:italic> aberrations (17p [del(17p)] and/or <jats:italic>TP53</jats:italic> mutation) were detected in 320/2332 patients (13.7%). Using NGS analysis, 429 <jats:italic>TP53</jats:italic> mutations were identified in 303 patients (13%). Of these 238 (79%) and 65 (21%) were cases with high burden and low burden mutations respectively. In our cohort, 2012 cases did not demonstrate a <jats:italic>TP53</jats:italic> aberration (86.3%). A total of 159 cases showed <jats:italic>TP53</jats:italic> mutations in the absence of del(17p) (49/159 with low burden <jats:italic>TP53</jats:italic> mutations) and 144 cases had both <jats:italic>TP53</jats:italic> mutation and del(17p) (16/144 with low burden mutations). Only 17/2332 (0.7%) cases demonstrated del(17p) with no <jats:italic>TP53</jats:italic> mutation. Validated NGS protocols should be used in clinical decision making to avoid missing low-burden <jats:italic>TP53</jats:italic> mutations and can detect the vast majority of <jats:italic>TP53</jats:italic> aberrations.</jats:p>
|
[
{
"section_content": "Deletion of chromosome 17p [del(17p)] and TP53 mutation (TP53 mut) referred to as TP53 aberrations can be found in 8%-10% of previously untreated chronic lymphocytic leukaemia (CLL) patients and in up to 30%-40% of relapsed/refractory cases.TP53 aberrations represent the most relevant risk factors for both progression free and overall survival following chemoimmunotherapy (1,2).The introduction of small molecule inhibitors has led to enhanced response rates in patients with TP53 aberrations (3)(4)(5).Therefore, the identification of TP53 aberrations is essential for determining treatment decisions in CLL (6,7).Historical data using Sanger sequencing suggests that approximately 80% of patients with del (17p) also carry a mutation in the second allele (8).A subset of patients also exhibits TP53 mut without del(17p) (8). The assessment of del(17p) is routinely performed by Fluorescence in situ hybridization (FISH).The cut-off for a positive result varies within laboratories with the threshold >20% of cells with del(17p) deemed to be a clinically relevant clone (9).However, it is recognized that a subset of patients with del(17p) have stable disease without the need for treatment (10). Sanger sequencing is widely used for TP53 mutational analysis, however it may misclassify cases of TP53 mutations as wildtype when variants with allelic frequencies below the detection limit of Sanger sequencing are present.Recent studies using next generation sequencing (NGS) have shown that TP53 mutations can be present at low clonal abundance in tumour cell populations, termed low-burden and have in certain studies the same detrimental effect on disease course (11)(12)(13).Therefore updated guidelines from the TP53 network of ERIC (European Research Initiative on CLL-www.ericcll.org)suggest a threshold of 10% allelic burden for reporting mutations detected by NGS segregating these into high burden (≥10 variant allele frequency (VAF)) and low burden (<10% VAF) mutation (6).In the literature, contradictory results exist regarding the biological relevance of low burden mutations in CLL.This in part may be due to various sequencing strategies. Therefore, the aim of this study, which is the largest cohort to date, was to investigate the presence of low and high burden TP53 mutations in a \"real-world\" cohort of 2332 CLL cases using sensitive NGS and to correlate results with FISH data. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "Pretreatment peripheral blood samples from 2332 CLL patients referred for analysis of del(17p13) by FISH and TP53 mutations by NGS were available for the present study diagnosed between 2015-2019.A retrospective audit of TP53 status was undertaken.Best practice in the UK follows established guidelines, meaning that TP53 testing is recommended prior to each line of treatment but not at diagnosis.As participants are part of Specialized HaemOnc Diagnostics services, requests for TP53 testing in newly diagnosed patients would automatically be rejected. The study was conducted according to the Declaration of Helsinki.Patients were diagnosed according to iwCLL guidelines (14).In all cases, analysis was performed on DNA obtained from >50% tumour cells.FISH analysis for del(17p13) was performed using Vysis Probes with a 10% cut-off for a positive result.TP53 mutation screening was performed by NGS with a panel covering exons 2-11 as previously described (15) or by an Illumina amplicon-based strategy.Briefly, the amplicon-based panel was a bespoke assay and amplicon libraries for are generated by Reverse Complement PCR (RC-PCR) technology.The technique permits both the amplification and the ability to append sequences or functional domains of choice independently to either end of the generated amplicons in a single closed tube reaction.Primers for the TP53 assay were designed in house and sequencing was performed on the Miseq using Illumina chemistry.Raw data was aligned using GATK.Indels are realigned using GeminiMulti indel realigner and Pisces is used for variant calling (both Illumina).Normally analyses with read depths below 5000 are failed. A VAF cut-off of 1% was used to exclude false positive variants within the cohorts.Pathogenicity assessment of all variants was performed according to ERIC guidelines (6). ",
"section_name": "METHODS",
"section_num": null
},
{
"section_content": "Altogether 2332 patients entering first line treatment were included in this study with TP53 aberrations detected in 320/ 2332 patients (Figures 1A,B).Using NGS analysis, 429 TP53 mutations were identified in 303 patients (13%).More than one TP53 mutation was detected in 76 patients (2-8 mutations per patient, Supplementary Table 1).When considering all 429 TP53 mutations in the cohort the VAF ranged from 1-97%; mean 28%.Using the 10% VAF threshold, cases were segregated into high burden mutations (≥10%) and low burden mutations (<10%).The high and low burden separation was based on the VAF of the most prevalent TP53 mutation.271 (63%) were classified as high burden mutations (VAF range: 10-97%; mean 42%).158 were classified as low burden mutations (VAF range: 1-9%; mean 5%) (Figure 2).This translated into 238 patients classified as high burden cases and 65 identified as low burden cases (Supplementary Table 1).The needle plot graphs demonstrated no differences in TP53 coding mutations between high and low burden cases (Figures 3A,B).The mutation profile revealed that the majority of mutations were missense mutations followed by frameshift, splicing and nonsense mutations and is in keeping with previous reports (Figure 3C) (16,17).No significant difference within mutation type existed between the low and high burden groups (P=0.5).The amino acid most frequently mutated were at positions 175, 209, 234, 248 and 273 indicating the classical hot spot mutations in CLL.Codons 175, 209, 234, 248 and 273 represented 110/429 (25%) mutations in the total cohort and showed similar allocation in low and high burden case (Figures 3A,B).Combining FISH data on del(17p) with TP53 mutation data in our cohort, 2012 cases did not demonstrate a TP53 aberration (86.3%).However, 17 cases demonstrated del(17p) only (0.7%).Average del(17p) was 40% (range 10-91%) in del(17p) only cases and was significantly higher in del(17p)/TP53 mut cases (55% (range 10-100%: p<0.05).One hundred and fifty-nine patients (159) were TP53 mutated only cases (49/159 with low burden TP53 mutations) and 144 cases with both del(17p) and TP53 mutation (16/144 with low burden mutations, Figure 1B). ",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "In this study, which is the largest study to date assessing TP53 aberrations for both del(17p) and TP53 mutation by NGS in cases of treatment naïve CLL.Using NGS analysis, 429 TP53 mutations were identified in 303 patients (13%).Current guidelines from the TP53 network of ERIC suggest a threshold of 10% allelic burden for reporting mutations detected by NGS ( 6).An acknowledgement is made in reference to cases with 5-10% VAF.In this study we employed a threshold of 10% VAF separating the cohort into high and low burden subgroups.High burden mutations were evident in 10.2% (238 cases) and low burden mutations in 2.8% (65 cases).This figure is lower than that reported in other studies and is likely due to the threshold of 1% used in this study (12,13).Even with this threshold, 49 cases [TP53 mut/del (17p) wt] in this cohort would have been misclassified as TP53 proficient cases.This is an important observation given the recent publication that clearly demonstrates a shorter survival in cases with VAFs of 5-10% (13).This study again questions the threshold of 10% VAF and the impact this has in the misclassification of TP53 aberrations. Whilst most tumour suppressors are inactivated by frameshift or nonsense mutations, the most frequent mode of inactivation of TP53 in CLL is by missense mutations which is a unique phenomenon.The mutation profile of the cohort did not differ when separated into high and low burden mutations.The vast majority of mutations were missense and no significant differences were observed between the low and high burden cohorts (Figures 3A-C).Unique to CLL is the presence of a specific hot spot variant leading to premature termination [p.(R209Kfs*6)].This specific variant was demonstrated both in low and high burden cases highlighting the similar mutation profile between the cohorts (Figures 3A,B).The majority of TP53 mutations are located within the DNA binding domain of the gene and hot spot mutations are frequently observed in CLL.This study showed an enrichment of mutations in codons 175, 209, 234, 248 and 273 representing (25%) of all mutations in the total cohort.A similar pattern was evident in both low and high burden subgroups confirming the disease specific TP53 mutation profile in CLL (Figures 3A,B) (16). Combining FISH data on del(17p) with TP53 mutation data in our cohort, 2012 cases did not demonstrate a TP53 aberration (86.3%) whereas TP53 aberrations were detected in 13.7% of patients.This is in keeping with recent data from independent groups that utilized various NGS strategies and bioinformatics pipelines (11,13,18). In this study we have demonstrated the existence of del(17p) in the absence of a TP53 mutation in 17/2332 (0.7%) which is in keeping with the literature (1,13).The average del(17p) clone was 40% with a range of 10-91% (Supplementary Table 1) with 8/17 cases having a del(17p) clone less than 25%.Patients in population based cohorts are still routinely screened for del(17p) by FISH, whilst testing for TP53 mutations can vary substantially by institution.This is despite very clear guidelines to the contrary (6,14).Screening for only del(17p) in our study would have missed 50% of the alterations in the cohort (159/320).The relevance of FISH only based studies in the era of NGS is questionable as only a minority of p53 deficient cases are missed by NGS.In this series 0.7% of p53 deficient cases were missed by NGS of which 8 cases had a del(17p) clone size of less than 25%.Also recent data showing low-frequency del(17p) sub clones (<25% of CLL cells) in the absence of a TP53 mutation has been demonstrated to mirror that of cases with no del(17p) in the chemoimmunotherapy setting (12,19).In the study by Do et al. 15/20 (75%) patients demonstrated a low frequency subclone of del(17p) (<25)).This is a well recognized phenomena in the literature with subset of patients with low frequency del(17p) clones having enhanced progression free survival (10).This subgroup of patients is enriched with a mutated IGHV gene and relatively few copy number alterations.The study by Do et al. represents a surprisingly high percentage of low level del (17p) not previously described and likely reflects the genomic composition of the elderly trial cohort in the study.In the current study, we demonstrated 28/144 (19%) cases where del(17p) <25% with 17 cases demonstrating a high burden (≥10%) mutations and 11 cases with low burden mutations (Figure 1B).Unfortunately clinical data was not available in this study to ascertain the IGHV status in the cohort of del(17p) subclones. TP53 aberrations are still relevant in the era of novel therapies.Long term survival outcomes remain inferior in cohorts of patients with TP53 aberrations (20,21).This is likely attributable to the role of p53 in the maintenance of genomic stability.It is well recognized that mutations in TP53 occur early in the disease progression proceeding the genomic instability generated by chromosomal abnormalities. This has been further addresses in a recent study demonstrating that patients treated with single-agent ibrutinib carrying only a single TP53 hit have a superior long term response while multi-hit TP53 is associated with a shorter progression free and overall survival (22).In this scenario single hit CLL can be classified by the presence of either del (17p) or TP53 mut.Multi hit CLL arises when either del(17p) and TP53 mut occur together or when greater than one TP53 mutation is found.Whilst this is of interest it has yet to be verified in larger cohorts or indeed in separate treatment regimens.In our current study 55% (176/320) were single hit with 45% (144/320) of cases demonstrating a multi hit CLL.In this study ≥2 TP53 mutations were detected in 76 patients with the majority of cases in the TP53 mut/del(17p) wt cohort (46/76) with the remaining 30 cases in the TP53 mut/no del(17p) cohort.This reinforces the need to redefine a VAF threshold to aid in the selection of TP53 mutated patients benefiting from targeted treatments. In conclusion, in the largest series to date we have demonstrated the presence of low and high burden TP53 mutations in a series of CLL cases.The use of NGS prevents cases being misclassified as normal TP53 due to its enhanced sensitivity.In the investigation of TP53 aberrations, NGS is an important strategy for patient management in this setting. ",
"section_name": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "The authors acknowledge Dr Karol Pal for his assistance in the preparation of figures. ",
"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/s. ",
"section_name": "DATA AVAILABILITY STATEMENT",
"section_num": null
},
{
"section_content": "Using the NHS Health Research Authority decision tools this study classes as clinical audit (i.e., no randomization of patients, no alteration to standard clinical care and informs practice in our setting) and therefore formal research ethics is not required. Author SL has received honoraria from Abbvie, Janssen, BMS/Celegene and Sanofi.DG has received honoraria, consultancy and/or research funding from Roche, AstraZeneca, Novartis, Janssen, Elli Lilly, Incyte, Promega and Illumina and is founder of Univ8 Genomics Ltd.Author NC has received honoraria from Novartis, Incyte and Astellas and research support from Novartis.Author AS received honoraria from Astra Zeneca, Janssen, Roche, Adaptive Biotechnology, Exact Sciences and AbbVie, and received nonrestricted educational grants from Astra Zeneca and Janssen and in-kind contributions from Illumina and Oxford Nanopore technologies.Author FF received honoraria from Abbvie, Janssen-cilag, Beigene, Astra-Zeneca and BC platform. The remaining 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": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "Using the NHS Health Research Authority decision tools this study classes as clinical audit (i.e., no randomization of patients, no alteration to standard clinical care and informs practice in our setting) and therefore formal research ethics is not required. ",
"section_name": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "Author SL has received honoraria from Abbvie, Janssen, BMS/Celegene and Sanofi.DG has received honoraria, consultancy and/or research funding from Roche, AstraZeneca, Novartis, Janssen, Elli Lilly, Incyte, Promega and Illumina and is founder of Univ8 Genomics Ltd.Author NC has received honoraria from Novartis, Incyte and Astellas and research support from Novartis.Author AS received honoraria from Astra Zeneca, Janssen, Roche, Adaptive Biotechnology, Exact Sciences and AbbVie, and received nonrestricted educational grants from Astra Zeneca and Janssen and in-kind contributions from Illumina and Oxford Nanopore technologies.Author FF received honoraria from Abbvie, Janssen-cilag, Beigene, Astra-Zeneca and BC platform. The remaining 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/s41408-022-00781-8
|
DNA-PK hyperactivation occurs in deletion 11q chronic lymphocytic leukemia and is both a biomarker and therapeutic target for drug-resistant disease
|
[
{
"section_content": "While chemoimmunotherapy has been the standard treatment for chronic lymphocytic leukemia (CLL), it has been supplanted by targeted therapies, including the Bruton's tyrosine kinase inhibitor, ibrutinib, and the bcl-2 inhibitor, venetoclax [1].The prognosis and prediction of response to chemotherapy are intimately linked to the leukemic cell IGHV mutational status and fluorescent in situ hybridization abnormalities [2].Patients with IGHV unmutated CLL have more aggressive disease with a preponderance of deletion 17p13 (del 17p; loss of TP53 gene) or del 11q22-23 (del 11q; loss of ataxia telangiectasia mutated (ATM) gene) cases. Del 11q occurs in 20% of CLL patients and is associated with a short time to first treatment (TTFT) and early relapse following chemotherapy [3].However, these patients have an excellent response to ibrutinib [4].Two-thirds of patients have an isolated del 11q and one-third an ATM mutation on the other allele, which can produce an additional loss of ATM function [5,6].However, an isolated del 11q has biological effects as telomere length is extremely short in del 11q cells, even without an additional ATM mutation [7].Moreover, the fraction of the cell population with del 11q is important, with shortening of TTFT only occurring when >25-58% of cells have the deletion [8,9].These data suggest that loss of one ATM gene can have biological effects. To determine if ATM protein levels are altered in CLL samples with del 11q, we examined the levels of total and phosphorylated (activated) ATM (pATM S1981 ) and DNA-PK (pDNA-PK S2056 ) by western blot in 30 patient samples with varying numbers of cells with del 11q (from 0 to 91%, Fig. 1A,B and Tables S1 andS2).Materials and Methods are in Supplementary.Twelve cases did not have del 11q or del 17 p (0%), 4 cases had a low del 11q fraction (<50%) while 14 cases had had a high del 11q fraction (>50%).While the levels of DNA-PK and ATM protein varied, ATM levels and the pATM:ATM ratio decreased as the fraction of del 11q cells increased (p = 0.009).These changes likely represent a dose-dependent loss of ATM activity as the fraction of cells with a del 11q increases, as mutations on the other allele do not necessarily correlate with further loss of ATM function [6].In contrast, the pDNA-PK:DNA-PK ratio increased as the fraction of del 11q cells increased (p = 0.023).Similarly, the ratio of activated DNA-PK relative to activated ATM (pDNA-PK:DNA-PK/pATM:ATM) increased significantly (p = 0.0001) as the fraction of del 11q cells (Fig. 1A-C and Table S2) increased.Thus, DNA-PK is activated in del 11q cases with a high fraction of del 11 cells, to compensate for loss of ATM. We then assessed the biological function of the DNA damage response (DDR) proteins in del 11q samples to determine if ATM function is lost and compensated for by an increase in DNA-PK activity.We thus compared non-del 11q and del 11q (>75% deletion) CLL samples following treatment with the DNA-crosslinking drug, chlorambucil, in the presence and absence of the DNA-PK inhibitor, NU7441.We also assessed the induction of phosphorylated KAP1 (pKAP1 S824 ) as a measure of ATM function [6].Chlorambucil increased DNA-PK activation to a 2.5-fold greater degree in del 11q samples, as compared to non-del 11q samples, and pDNA-PK formation was eliminated by NU7441 (Figs. 1D,E and S1 and Tables S1 andS3).pATM and pKAP1 levels were similarly increased by chlorambucil in all cases.pATM and pKAP1 increased further in non-del 11q samples following NU7441 but not in del 11q cells. To evaluate the DDR, we compared the effects of NU7441 by γH2AX foci formation (measures DDR signaling at breaksites) and alkali comet assay (measures DNA strand breaks) in irradiated samples [10].Del 11q cells (>50%) developed lower γH2AX levels compared to non-del 11q cells following irradiation (Fig. 1F and Table S1).Co-treatment with NU7441 delayed γH2AX formation and clearance, especially in del 11q samples (Fig. 1G and Table S1).Although ATM kinase is typically involved in γH2AX formation, DNA-PK is invoked with sub-optimal ATM function [11].Furthermore, DNA strand break repair did not appear to be affected by del 11q status or DNA-PK activity (Fig. S2 and Table S1).Thus, ATM mediates DDR signaling in non-del 11q samples but these activities may be replaced by DNA-PK in del 11q cells [11]. DNA-PK activity may also be increased in CLL patients who have received prior chlorambucil [12].We thus examined DNA-PK and ATM activation in six patients who received chemoimmunotherapy and then ibrutinib following relapse (Fig. 1H and Table S4).Activation of DNA-PK was significantly increased in four patients relapsing after chemoimmunotherapy (45, 708, 929, 322) paralleled by a decrease in activated ATM.In contrast, two patients had low pDNA-PK prior to therapy with little change following chemotherapy.DNA-PK activation increased further following ibrutinib, with a decline in activated ATM. In contrast to ATM, enhanced DNA-PK supresses homologous recombination, induces non-homologous end-joining (NHEJ) and is associated with aggressive disease in multiple cancers [13].To determine if this was true in CLL, pDNA-PK levels were compared in 16 new CLL patients, 8 requiring therapy in a median of 3 years, while 8 did not require treatment in this time period (Fig. 1I and Table S5).The baseline pDNA-PK:DNA-PK level was higher in those that required therapy suggesting that increased DNA-PK may explain the short TTFT in del 11q (p = 0.015). To determine whether the elevated DNA-PK activity in del 11q cells influenced drug sensitivity, CLL samples were treated with NU7441.Interestingly, del 11q CLL cells were more sensitive to NU7441 than non-del 11q cases [14].In total, 1 µM NU7441 decreased cell viability by 30% at 72 h (Fig. 2A and Table S1).In contrast, NU7441 had little activity against normal B (CD19+) and T (CD3+) cells, demonstrating that this is a tumor specific activity. Inhibition of DNA-PK has been shown to enhance drug sensitivity in CLL, so we assessed whether this was greater in del 11q cells (Fig. 2B,C and Tables S1 andS6) [14].Six samples had >50% del 11q, 4 < 50% del 11q, 17 were non-del 11q and 1 del 17p.Following 72 h treatment, cell death was measured by Annexin V/7AAD staining and the drug concentration required to reduce cell viability by 50% (EC 50 ) was measured.Samples with >50% del 11q cells were more resistant to chemotherapy than non-del 11q patients (median chlorambucil EC 50 , 38.1 μM versus 15.8 μM for del 11q and non-del 11q, respectively; Fig. 2B, p = 0.013).In contrast, the median ibrutinib EC 50 for del 11q and non-del 11q cells were similar, at 6.1 and 3.9 μM, respectively (Fig. 2C, p = 0.3124).However, NU7441 sensitized both del 11q and non-del 11q cells to the chemotherapies chlorambucil, fludarabine or bendamustine to a similar extent (Tables S1 andS6).Resistant cells were also sensitized, including a sample with del 17p.NU7441 did not sensitize CLL cells to ibrutinib or idelalisib.NU7441 produced little sensitization of normal B and T cells to chlorambucil (Tables S1 andS6). As NU7441 is not for clinical use, we evaluated M3814 and CC-115 in CLL cells, as both agents are being evaluated clinically [13,15].Initially, CLL cells from two patients were treated with CC-115 (inhibitor of DNA-PK/mTOR/PI3K) or M3814 (inhibitor of DNA-PK; Fig. S3) and chlorambucil or ibrutinib.Synergy was greater between chlorambucil and M3814 than between chlorambucil and CC-115; however, ibrutinib showed synergy with CC-115 but not M3814 (Fig. S4), likely a result of the effect of CC-115 on mTOR/PI3K activity.Further studies with M3814 showed it to be strongly synergistic with chlorambucil in CLL cells from 14 untreated patients without del 17p (Figs.2D andS5), independent of del 11q status or anti-IgM treatment. In patients on long-term ibrutinib, we observed enhanced pDNA-PK levels over time, which inversely correlated with pATM levels (Fig. 2E and Table S7).Importantly, sensitization between chlorambucil and M3814 was observed in cells from three of five patients on long-term ibrutinib (505, 190, and 847) including one (505) who was resistant to ibrutinib (Fig. 2D,F).Synergy increased throughout 1 year of ibrutinib treatment in patient 190 (Fig. S3).The combination of ibrutinib with M3814 in cells from patients on long-term ibrutinib showed additive or slightly synergistic cytotoxicity in three of four patients (505, 830, and 847) while the del 17p sample (171) showed antagonism (Fig. 2D,F). These studies show that DNA-PK is activated and responsible for DDR in del 11q CLL cells, and this may explain the short TTFT and resistance to chemotherapy, but not ibrutinib, in these patients.DNA-PK is not activated by disease duration or progression but increases following ibrutinib and chemotherapy, suggesting that it is an effect of the DNA damage signaling response/NHEJ.Del 11q cells were more sensitive to DNA-PK inhibition than non-del 11q cells or normal lymphocytes, but inhibition sensitized all CLL types (but not normal lymphocytes) to chemotherapy, including those that were both chemotherapy-and ibrutinib-resistant.Hyperactivated DNA-PK is associated with aggressive disease in a variety of hematologic and solid tumors; likely reflecting enhanced mutation-prone NHEJ activity with genomic instability [13].Thus, combining M3814 with chemotherapy may be a useful approach for treating multidrug-resistant CLL patients. Fig. 1 Impact of an increasing fraction of del 11q CLL cells on DNA-PK and ATM activation.A Western blot demonstrating the effect of a varying percentage of del 11 cells on protein levels of ATM, DNA-PK, and their activated phosphorylated derivatives, pDNA-PK and pATM.B Levels of DNA-PK, ATM, pATM:ATM, pDNA-PK:DNA-PK and pDNA-PK:DNA-PK/pATM:ATM in del 11q and non-del 11q cells (p values calculated using a Mann-Whitney test) and C relationship between the percentage of CLL cells with a deletion (del) 11q and the levels of DNA-PK, ATM and their phosphorylated derivatives (p values calculated using linear regression).Samples were all from untreated patients within 6 months of their FISH analysis.Vinc vinculin.Influence of chlorambucil and NU7441 treatment on DNA-PK activation and ATM function in del 11q CLL cells.D Representative western blots for (E) densitometry following ~18 h treatment of primary CLL cells in serum free media (SFM) or with CD40L/IL4 showing an increase in pDNA-PK in del 11q cells following 15 μM chlorambucil (CLB) treatment that was eliminated by combination with 1 μM NU7441 (NU), whereas pATM and pKAP1 levels were reduced in the del 11q cells with this cotreatment.Bars represent phospho-protein, dots represent total protein.Impact of DNA-PK inhibition γH2AX formation in del 11q CLL cells.CLL cells were treated for ~18 h with DMSO or 1 μM NU7441 in SFM alone or in the presence of CD40L/IL4 and irradiated during drug treatment with (F) 20 Gy or (G) 5 Gy and allowed to recover for 3, 1, or 0 h or 18, 6, or 0 h, respectively.Analyses of serine 139 phosphorylation of alternative histone H2AX (γH2AX) MFI (median fluorescence intensity) reveals that del 11q CLL cells accumulate less γH2AX compared to non-del 11q counterparts following IR-mediated induction of DNA strand breaks.While NU7441 co-treatment delays γH2AX formation in both CLL subtypes, del 11q cells are affected to a greater degree.DNA damage response (DDR) and B cell receptor (BCR) kinase activation in CLL patients following chemoimmunotherapy and short term ibrutinib treatment.H Western analysis of primary patient-matched CLL cells prior to treatment (P), following fludarabine, cyclophosphamide, rituximab (FCR) or chlorambucil/ obinutuzumab chemotherapy (C) and four patient samples on ibrutinib (I; 45, 708, 929 and 322).Ratio of pDNA-PK:DNA-PK shows a progressive treatment-dependent increase of activated DNA-PK following chemotherapy and ibrutinib, coinciding with concomitantlyreduced ATM activation (pATM:ATM) and ibrutinib-mediated reduction in BTK activity (pBTK:BTK).Enhanced basal DNA-PK activation indicates eventual treatment.I Western analysis derived from these primary CLL patient cells shows that amongst patients who ultimately progressed to treatment (Tx), activated DNA-PK levels at diagnosis were significantly higher compared to those who did not require treatment after a similar time frame, while total DNA-PK levels were similar amongst the two cohorts.All plots in this figure are median ± interquartile range. ",
"section_name": "Dear Editor,",
"section_num": null
}
] |
[
{
"section_content": "The Manitoba Tumor Bank (Winnipeg, Manitoba) is supported by the CancerCare Manitoba Foundation and is a member of the Canadian Tissue Repository Network.We are thankful to Drs.Jody Haigh (U.Manitoba) and Roseline Godbout (U.Alberta) for their comprehensive pre-submission review of the final manuscript. ",
"section_name": "ACKNOWLEDGEMENTS",
"section_num": null
},
{
"section_content": "Supported by Research Manitoba (Manitoba CLL Cluster Grant), Canadian Institutes of Health Research, Canadian Foundation for Innovation, and CancerCare Manitoba Foundation. ",
"section_name": "FUNDING",
"section_num": null
},
{
"section_content": "SEFK, AS, SHY, and LY carried out the experiments with technical assistance from BK. SEFK, SBG, JBJ, and SK designed the research, collected, analyzed and interpreted data.VB, LY, and JBJ cared for patients contributing samples.SEFK, JBJ, and SK wrote the manuscript.All authors approved the manuscript in its final format. VB and LY: advisory board-members for Abbvie, Janssen and AstraZeneca. Fig. 2 NU7441 is cytotoxic to CLL cells, but not normal B/T cells, and sensitizes CLL patient cells to chemotherapy.Primary CLL cells or PBMCs from non-CLL donors were treated with chlorambucil (CLB) or ibrutinib (IBR) for ~72 h alone or in the presence of 1 μM NU7441 (NU) in SFM, or co-treated with CD40L/IL4 or IgM and viability was measured by flow cytometry.A While CLL cell viability is reduced by NU7441, normal B (CD19+) and T (CD3+) cells are not.B Both del 11q and non-del 11q CLL cells treated with chlorambucil result in multi-fold lower EC 50 values when co-treated with NU7441, while (C) EC 50 values of ibrutinib are not affected by NU7441 co-treatment.Plots represent median ± interquartile range and p values were calculated using a paired t-test or a Mann-Whitney test.DNA-PK inhibition is synergistic with chlorambucil, even in CLL cells from patients on developing resistance to ibrutinib.D Combenefit synergy plots from CLL samples from patients who were treatment (Tx) naïve or on long-term ibrutinib treated ex vivo for ~72 h with combinations of chlorambucil or ibrutinib with M3814 in SFM, demonstrating marked synergy between M3814 and chlorambucil but not ibrutinib, even in patients who were receiving ibrutinib or were resistant to ibrutinib.Plots represent the degree of synergy (blue), additivity (green), or antagonism (red).Changes in DNA-PK and ATM activation are inversely correlated while CLL patients are on long-term ibrutinib.E Western blot of CLL cells taken prior to and while on long-term ibrutinib.pDNA-PK:DNA-PK levels increase in most patients while on long-term ibrutinib, pATM:ATM levels decrease in some patients and pBTK:BTK is eliminated by ibrutinib.DNA-PK inhibition is synergistic with chlorambucil, but not ibrutinib, in CLL patient cells following long-term ibrutinib treatment or resistance.F EC 50 of chlorambucil and ibrutinib determined by flow cytometry (from 2D) following ~72 h treatment alone or with 1 μM NU7441 or 250 nM M3814 before or during clinical treatment of the patient, showing sensitization of cells to chlorambucil but not ibrutinib even when patients were receiving chlorambucil (1020), ibrutinib (733,1440,190,171) or were refractory to ibrutinib (505, 830).Acala acalabrutinib. The online version contains supplementary material available at https://doi.org/10.1038/s41408-022-00781-8. Correspondence and requests for materials should be addressed to James B. Johnston or Sachin Katyal. 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": "SEFK, AS, SHY, and LY carried out the experiments with technical assistance from BK. SEFK, SBG, JBJ, and SK designed the research, collected, analyzed and interpreted data.VB, LY, and JBJ cared for patients contributing samples.SEFK, JBJ, and SK wrote the manuscript.All authors approved the manuscript in its final format. ",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "VB and LY: advisory board-members for Abbvie, Janssen and AstraZeneca. Fig. 2 NU7441 is cytotoxic to CLL cells, but not normal B/T cells, and sensitizes CLL patient cells to chemotherapy.Primary CLL cells or PBMCs from non-CLL donors were treated with chlorambucil (CLB) or ibrutinib (IBR) for ~72 h alone or in the presence of 1 μM NU7441 (NU) in SFM, or co-treated with CD40L/IL4 or IgM and viability was measured by flow cytometry.A While CLL cell viability is reduced by NU7441, normal B (CD19+) and T (CD3+) cells are not.B Both del 11q and non-del 11q CLL cells treated with chlorambucil result in multi-fold lower EC 50 values when co-treated with NU7441, while (C) EC 50 values of ibrutinib are not affected by NU7441 co-treatment.Plots represent median ± interquartile range and p values were calculated using a paired t-test or a Mann-Whitney test.DNA-PK inhibition is synergistic with chlorambucil, even in CLL cells from patients on developing resistance to ibrutinib.D Combenefit synergy plots from CLL samples from patients who were treatment (Tx) naïve or on long-term ibrutinib treated ex vivo for ~72 h with combinations of chlorambucil or ibrutinib with M3814 in SFM, demonstrating marked synergy between M3814 and chlorambucil but not ibrutinib, even in patients who were receiving ibrutinib or were resistant to ibrutinib.Plots represent the degree of synergy (blue), additivity (green), or antagonism (red).Changes in DNA-PK and ATM activation are inversely correlated while CLL patients are on long-term ibrutinib.E Western blot of CLL cells taken prior to and while on long-term ibrutinib.pDNA-PK:DNA-PK levels increase in most patients while on long-term ibrutinib, pATM:ATM levels decrease in some patients and pBTK:BTK is eliminated by ibrutinib.DNA-PK inhibition is synergistic with chlorambucil, but not ibrutinib, in CLL patient cells following long-term ibrutinib treatment or resistance.F EC 50 of chlorambucil and ibrutinib determined by flow cytometry (from 2D) following ~72 h treatment alone or with 1 μM NU7441 or 250 nM M3814 before or during clinical treatment of the patient, showing sensitization of cells to chlorambucil but not ibrutinib even when patients were receiving chlorambucil (1020), ibrutinib (733,1440,190,171) or were refractory to ibrutinib (505, 830).Acala acalabrutinib. ",
"section_name": "CONFLICT OF INTEREST",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https://doi.org/10.1038/s41408-022-00781-8. Correspondence and requests for materials should be addressed to James B. Johnston or Sachin Katyal. 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
}
] |
|
10.21608/resoncol.2021.51731.1128
|
COVID-19 Infection in Patients with Chronic Lymphocytic Leukemia: Report of Two Cases and Literature Review
|
Chronic lymphocytic leukemia (CLL) typically occurs in the elderly and has a highly variable clinical course. Infectious complications have been known to be a major cause of morbidity and mortality in CLL patients. The management of hematological malignancies, including CLL, during the COVID-19 pandemic is challenging. Here we describe two patients with CLL who got infected with SARS-COV-2 as confirmed by positive nasopharyngeal swab PCR. The 1st patient, who was receiving treatment for CLL-associated autoimmune hemolytic anemia, was hospitalized with mild COVID-19 symptoms. The 2nd patient, who was on active treatment for CLL, had asymptomatic COVID-19 infection and was not hospitalized. Both patients recovered from COVID-19 without related complications.
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is characterized by the clonal proliferation and accumulation of mature, typically CD5-positive B cells within the blood, bone marrow, lymph nodes, and spleen.It has been reported that the capacity to generate clonal B cells in CLL may be acquired at the hematopoietic stem cell stage 1 . In general, patients with CLL are considered to be at high-risk for infections, mainly bacterial or infections of the herpes virus family (e.g., herpes simplex virus and varicella-zoster virus).This is due to an underlying immunodeficiency, inadequate immune response to infections and therapy-related immunosuppression 2 .The immune dysfunction in patients with CLL raises concern and represents a challenge during the COVID-19 pandemic 3 . During the current COVID-19 pandemic, CLL patients are theoretically at higher risk of contracting coronavirus 2 (SARS-COV-2) infections and are expected to experience more severe symptoms if they are older.However, at this time, there is no evidence indicating a disproportionately higher incidence of severe COVID-19 in patients with CLL compared to patients with other malignancies 2 .A systematic review with meta-analysis showed that the risk of mortality among SARS-COV-2 infected CLL patients is lower than that in other hematological malignancies 4 . Herein, we discuss two cases of CLL who contracted and recovered from COVID-19 infection without serious complications. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "The characteristics of the two reported cases and the summary of CLL and COVID-19 infection data are shown in Table 1. ",
"section_name": "Case Presentation",
"section_num": null
},
{
"section_content": "A 64-year-old Saudi male known to have hypertension and chronic renal disease was referred to Aseer Central Hospital with leukocytosis in March 2020. He seemed well, but complaining of chest pain radiating to the left shoulder, later diagnosed as acute coronary syndrome.Examination showed bilateral small cervical lymphadenopathy with splenomegaly 2-3 fingers below the costal margin. The initial laboratory work-up is shown in Table 1.Peripheral smear and flow cytometry analysis of the peripheral blood showed the presence of abnormal B lymphocytes population gated at CD45 dim to bright.These abnormal population was positive for CD19, CD5, CD20 (dim), CD22 (dim), CD23, CD38 (dim), CD79b (dim) and BCL2 (dim) with kappa light chain restriction.The gated cells were negative for FMC7, CD3, CD4, CD8, CD10 and CD34.The picture was consistent with B-cell CLL. Pelvic-abdominal ultrasound revealed splenomegaly and chest X-ray showed no mediastinal lymph nodes.Hence, the patient was finally diagnosed as CLL, Rai stage II.According to the iwCLL guidelines 5 , the patient was a candidate to receive treatment for auto-immune hemolytic anemia (AIHA) only. As regards the acute coronary syndrome, the patient received antiplatelet as well as thrombolytic therapy.This was complicated by an intracranial hemorrhage and he was admitted to the intensive care unit for 15 days.Concomitantly, treatment for AIHA was started with steroids and rituximab. One month after discharge from the intensive care unit and while he was receiving treatment for AIHA, he developed a fever (38.5 o ), flu-like symptoms and bony pains.He sought medical advice at a local hospital where he was admitted and tested positive for SARS-COV-2 by nasopharyngeal swab PCR.Serum ferritin, lactate dehydrogenase and Ddimer levels were within the normal ranges as well as liver function tests and biochemistry.Complete blood picture showed mild anemia with absolute lymphocytosis (Table 1).Chest X-ray revealed no pulmonary infiltration. The patient received treatment in the form of zinc, antipyretics, azithromycin, and ceftriaxone.His symptoms improved after two days.Treatment for AIHA was continued after clinical improvement without waiting a negative swab.The swab result was negative twice with a 72h interval on the 1 st and 3 rd August 2020.The patient recovered from COVID-19 infection without complications and did not need intravenous Ig. ",
"section_name": "Case No. 1",
"section_num": null
},
{
"section_content": "A 66-year-old Palestinian male, known to have diabetes mellitus, hypertension, and chronic renal disease, had been diagnosed with CLL stage II in August 2018.His initial laboratory work-up is shown in Table 1.Peripheral smear and immunophenotyping results were consistent with CLL.As per the iwCLL guidelines, there was no indication to treat CLL and a watch and wait strategy was followed.In March 2020, he presented with weight loss, night sweats and fever and splenomegaly reaching the umbilicus.The CBC showed a white blood cell count of 213 *103/µL, hemoglobin 8.9 gm/dL, and platelet count 109 *103/µL.Post-contrast Computed tomography showed generalized lymphadenopathy, hepatosplenomegaly and multinodular goiter with.The patient started treatment with FCR (fludarabine, cyclophosphamide and rituximab) regimen on 9 March 2020 with a very good partial response after the 3 rd cycle.The 4 th cycle was administered on 2 June 2020. Two weeks after the administration of the 4 th FCR cycle, one family member who had been in close contact with the patient tested positive for COVID-19.Accordingly, the patient underwent a PCR nasopharyngeal swabs for SARS-COV-2 which was found to be positive.The patient continued antiviral and antibacterial prophylaxis which was part of the CLL treatment protocol.D-dimer and c-reactive protein levels were within the normal ranges, while lactate dehydrogenase was above normal.The CBC result is shown in Table 1 and liver and kidney function tests were normal.The second swab after 15 days was also positive.When a negative swab was detected, FCR was resumed after a delay of 50 days in receiving chemotherapy.The patient was doing well without any COVID-19-related complications. ",
"section_name": "Case No. 2",
"section_num": null
},
{
"section_content": "As of the end of January 2021, the pandemic of COVID-19 caused by SARS-CoV-2 has already affected over 102 million persons and caused more than 2.2 million confirmed deaths around the world 6 .Infection with SARS-CoV-2 causes critical disease in approximately 5% of affected patients, particularly those who have risk factors such as cancer, older age, immunodeficiency (hematological cancer, including CLL), or such chronic diseases as diabetes mellitus and COPD 7,8 .It is known that infection is the most common complication and the first cause of death in CLL.The belief that patients with cancer are at high risk of contracting COVID-19 derives from reports describing the underlying conditions in patients with COVID-19 7,9 .The pathogenesis of infection in CLL is multifactorial, with alterations due to the primary disease process and immunosuppression caused by subsequent treatments; however, A study found that cellular immunotherapy impeded CLL, which may be related to the acquired immune dysfunction that mainly manifests as the abnormal expansion of T cells, failure to form synaptic T cells, and inhibition of T cell migration [10][11][12] . As many studies published about CLL patients who contracted COVID-19 are shown in Table 2.The studies show how this type of viral infection presented with severe symptoms and a poor outcome in both naïve CLL patients without treatment and patients on active treatment.Therefore, clinicians should act with particular caution in relation to those with CLL who contract COVID-19, because most of them are elderly and have added co-morbidities.Ye et al, 2020 19 Case report 1 72 Langerbeins et al, 2020 20 Case report 1 52 Favresse et al, 2020 21 Case report 2 N/A • Hydroxychloroquine 2 0 As regards our patients, contrary to expectation, they experienced and recovered from COVID-10 without problems.Although our patients had additional risk factors (hypertension, diabetes mellitus, cardiovascular disease, chronic kidney disease), the first patient had mild symptoms, while the second did not experience any symptoms while he was on active treatment with an FCR regimen.The first case was discharged three days after admission.Both were put under home quarantine, with scheduled regular follow-up telephone visits. The experience of the second patient ran counter to what has been shown by Furstenau et al 12 in a small case series, where the most severe respiratory failures were observed in patients who were still under treatment or had stopped treatment two months before. A retrospective international multicenter study done by Scarfò et al 13 demonstrated the course and severity of COVID-19 in patients with CLL, in which 151(79%) out of a total 190 patients who contracted COVID-19 presented with severe disease (needing oxygen and/or admission to intensive care).Of the 151 patients with severe disease, 55 (36.4%) died.The hospitalization rate for severe COVID-19 was lower (p<0.05)for patients on ibrutinib than for those on other regimens or off treatment.This supports the idea that antileukemic treatment (particularly BTK inhibitors) appears to exert a protective effect against the coronavirus.As treatment may predispose to greater immunodeficiency as well as more severe infection and poor outcome, treatment should be delayed in patients who are oligosymptomatic and those with non-life-threatening cytopenias 14 . On other hand, the study of Mato et al 15 on CLL patients (n=198) diagnosed with symptomatic COVID-19 across 43 international centers revealed that hospital admission occurred in 90%.Thirty-nine percent were treatment-naïve, while 61% had received ≥1 dose of CLL-directed therapy.\"Watch and wait\" and treated cohorts had similar rates of admission (89% vs. 90%), ICU admission (35% vs. 36%), intubation (33% vs.25%), and mortality (37% vs. 32%).CLL-directed treatment with BTKi at COVID-19 diagnosis did not impact survival (CFR 34% vs.35%).These data suggest that the subgroup of CLL patients admitted with COVID-19, regardless of disease phase or treatment status, are at high risk of death. Case No.2 in the present study was on antiviral prophylaxis (acyclovir 400 mg BID) with antibacterial prophylaxis sulfamethoxazole /trimethoprim.This raises the question whether antiviral prophylaxis could be protective against COVID-19 or prevent severe symptoms.Case No.1 was therapy-naïve, having just received steroids and two doses of rituximab to treat autoimmune hemolytic anemia (AIHA) before contracting COVID-19.CBC during infection did not show any increase in the lymphocyte count, which is contrary to the observations of Paneesha et al 16 , who documented four patients with untreated CLL who contracted SARS-CoV-2.Their clinical pathway was severe, carrying a high rate of mortality.In addition, an increase in the lymphocyte count, a phenomenon termed 'COVID-induced lymphocytosis', was observed in all cases with an average of three-fold increase during COVID-19. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "The two cases discussed here show that some CLL patients may experience COVID-19 with no major problems.There is a need for more studies on the management of CLL and other hematological malignancies during the COVID-19 pandemic. ",
"section_name": "Conclusion",
"section_num": null
}
] |
[
{
"section_content": "None. ",
"section_name": "Acknowledgment",
"section_num": null
},
{
"section_content": "Deidentified individual participant data used to produce the results of this study are included in this published article. ",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "The authors declare that they have no conflict of interest to disclose. This case report was approved in December 2020 by the Ethics and IRB (Internal Review Board) Committee of Aseer Central Hospital, Abha, Saudi Arabia.A verbal consent was obtained from the patients due to COVID-19 limitations. The authors did not receive funding for this study. None. ",
"section_name": "Authors' contribution",
"section_num": null
},
{
"section_content": "",
"section_name": "Authors' contribution",
"section_num": null
},
{
"section_content": "The authors declare that they have no conflict of interest to disclose. ",
"section_name": "Conflict of interest",
"section_num": null
},
{
"section_content": "This case report was approved in December 2020 by the Ethics and IRB (Internal Review Board) Committee of Aseer Central Hospital, Abha, Saudi Arabia.A verbal consent was obtained from the patients due to COVID-19 limitations. ",
"section_name": "Ethical Considerations",
"section_num": null
},
{
"section_content": "The authors did not receive funding for this study. ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "None. ",
"section_name": "Study registration",
"section_num": null
}
] |
10.18632/aging.100811
|
Inhibition of notch promotes liver metastasis
|
Classically known for its role in development and differentiation, an oncogenic role for Notch was first discovered for T-cell acute lymphoblastic leukemia [1], and later extended to a variety of tumors [2]. Notch signaling has been mechanistically linked to regulate tumor growth, angiogenesis and metastatic progression. Consequently, in the last decade we have witnessed multiple therapeutic approaches to target Notch, including γ-secretase inhibitors (GSIs), which block cleavage and activation of Notch receptors, soluble Notch decoy receptors, and antibodies to either Notch receptors or ligands [3]. However due to widespread function and highly pleotropic nature, Notch inhibition raises the possibility of unanticipated adverse effects in host tissues including gastrointestinal toxicity after GSI treatment or formation of vascular tumors after DLL4 ligand blockade [4, 5]. Our current work published in the journal Cancer Research, has raised one such concern about Notch inhibition therapy [6]. Inhibition of Notch with the soluble receptor Notch1 decoy (N1D), markedly increased liver metastatic burden, of neuroblastoma and breast cancer cells lines after intrarenally or intracardiac injection (spontaneous vs. experimental metastasis models). Additionally, pharmacologic inhibition of Notch with the GSI PF-03084014, while having no effect on primary tumor growth, significantly increased liver metastases. Interestingly, we did not observe in any of the experimental models, an increase in metastasis to other organs including spleen, kidney and bone marrow, suggesting that Notch blockade specifically promotes metastases to the liver. Our studies determined that the increased liver metastases are a result of a direct effect of Notch inhibition on host liver, with the loss of tumor cell-intrinsic Notch1 signaling inconsequential. Tumor cells expressing N1D showed no change in prometastatic characteristics such as migration and invasion. Loss of Notch signaling in tumor cells by knocking down Notch1 also failed to promote liver metastases. However, intracardiac injection of tumor cells into immunodeficient (Rag2−/−, Il2rg−/−) Notch1 +/− mice, where host Notch signaling was deficient due to heterozygous deletion of Notch, demonstrated a marked increase in hepatic metastases, indicating that Notch1 signaling acts as metastatic suppressor in the liver microenvironment. The liver microenvironment is composed of a variety of cell types, including sinusoidal endothelial cells (SECs), hepatic stellate cells (HSCs), and macrophage Kupffer cells, which express Notch receptors and ligands [7]. One striking characteristic of the liver metastases promoted by Notch blockade was their markedly increased vascularity. We observed a significant increase in the sprouting of hepatic SECs into early stage, micrometastases. Utilizing Notch decoy variants that are specific for the Notch ligands DLL and JAGGED, we determined that the increased sprouting was due to blockade of DLL on SECs. In the later stage, macrometastases, there was a marked increase in larger diameter vasculature that was α-smooth muscle actin (+), indicating activation and recruitment of HSCs into the vasculature. This was likely due to the loss of tumor derived JAG1, as indicated by blockade with a JAGGED specific decoy. Our data support a concept of Notch signaling maintaining the quiescence of SECs via DLL and HSCs via JAG1, with disruption of this quiescence promoting a prometastatic environment. Figure 1 Notch inhibition activates sinusoidal endothelial cells and hepatic stellate cells to promote hepatic metastases. Our study was primarily focused on Notch1, however, it would be interesting to investigate if other Notch receptors have similar roles since they are reported to regulate SEC and HSC function to maintain liver home-ostasis [7]. Given that metastasis progression is a multistep process it would also be interesting to investigate if Notch signaling regulates other steps including survival against host innate immunity. Nonetheless, our study shows that Notch inhibition results in pathological activation of liver stromal cells creating a host microenvironment favorable for metastases. Given that Notch components have emerged as potential therapeutic targets and Notch inhibitors are entering clinical trials these findings point out the potentially serious implication of Notch inhibition therapy.
|
[
{
"section_content": "Classically known for its role in development and differentiation, an oncogenic role for Notch was first discovered for T-cell acute lymphoblastic leukemia [1], and later extended to a variety of tumors [2].Notch signaling has been mechanistically linked to regulate tumor growth, angiogenesis and metastatic progression.Consequently, in the last decade we have witnessed multiple therapeutic approaches to target Notch, including γ-secretase inhibitors (GSIs), which block cleavage and activation of Notch receptors, soluble Notch decoy receptors, and antibodies to either Notch receptors or ligands [3].However due to widespread function and highly pleotropic nature, Notch inhibition raises the possibility of unanticipated adverse effects in host tissues including gastrointestinal toxicity after GSI treatment or formation of vascular tumors after DLL4 ligand blockade [4,5].Our current work published in the journal Cancer Research, has raised one such concern about Notch inhibition therapy [6].Inhibition of Notch with the soluble receptor Notch1 decoy (N1D), markedly increased liver metastatic burden, of neuroblastoma and breast cancer cells lines after intrarenally or intracardiac injection (spontaneous vs. experimental metastasis models).Additionally, pharmacologic inhibition of Notch with the GSI PF-03084014, while having no effect on primary tumor growth, significantly increased liver metastases.Interestingly, we did not observe in any of the experimental models, an increase in metastasis to other organs including spleen, kidney and bone marrow, suggesting that Notch blockade specifically promotes metastases to the liver.Our studies determined that the increased liver metastases are a result of a direct effect of Notch inhibition on host liver, with the loss of tumor cellintrinsic Notch1 signaling inconsequential.Tumor cells expressing N1D showed no change in prometastatic characteristics such as migration and invasion.Loss of Notch signaling in tumor cells by knocking down Notch1 also failed to promote liver metastases.However, intracardiac injection of tumor cells into immunodeficient (Rag2-/-, Il2rg-/-) Notch1 +/-mice, where host Notch signaling was deficient due to heterozygous deletion of Notch, demonstrated a marked increase in hepatic metastases, indicating that Notch1 Editorial signaling acts as metastatic suppressor in the liver microenvironment.The liver microenvironment is composed of a variety of cell types, including sinusoidal endothelial cells (SECs), hepatic stellate cells (HSCs), and macrophage Kupffer cells, which express Notch receptors and ligands [7].One striking characteristic of the liver metastases promoted by Notch blockade was their markedly increased vascularity.We observed a significant increase in the sprouting of hepatic SECs into early stage, micrometastases.Utilizing Notch decoy variants that are specific for the Notch ligands DLL and JAGGED, we determined that the increased sprouting was due to blockade of DLL on SECs.In the later stage, macrometastases, there was a marked increase in larger diameter vasculature that was α-smooth muscle actin (+), indicating activation and recruitment of HSCs into the vasculature.This was likely due to the loss of tumor derived JAG1, as indicated by blockade with a JAGGED specific decoy.Our data support a concept of Notch signaling maintaining the quiescence of SECs via DLL and HSCs via JAG1, with disruption of this quiescence promoting a prometastatic environment. Our study was primarily focused on Notch1, however, it would be interesting to investigate if other Notch receptors have similar roles since they are reported to regulate SEC and HSC function to maintain liver home- ostasis [7].Given that metastasis progression is a multistep process it would also be interesting to investigate if Notch signaling regulates other steps including survival against host innate immunity.Nonetheless, our study shows that Notch inhibition results in pathological activation of liver stromal cells creating a host microenvironment favorable for metastases.Given that Notch components have emerged as potential therapeutic targets and Notch inhibitors are entering clinical trials these findings point out the potentially serious implication of Notch inhibition therapy. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Inhibition of",
"section_num": null
}
] |
[] |
10.1371/journal.pone.0038811
|
Inhibition of Fibroblast Growth by Notch1 Signaling Is Mediated by Induction of Wnt11-Dependent WISP-1
|
Fibroblasts are an integral component of stroma and important source of growth factors and extracellular matrix (ECM). They play a prominent role in maintaining tissue homeostasis and in wound healing and tumor growth. Notch signaling regulates biological function in a variety of cells. To elucidate the physiological function of Notch signaling in fibroblasts, we ablated Notch1 in mouse (Notch1(Flox/Flox)) embryonic fibroblasts (MEFs). Notch1-deficient (Notch1(-/-)) MEFs displayed faster growth and motility rate compared to Notch1(Flox/Flox) MEFs. Such phenotypic changes, however, were reversible by reconstitution of Notch1 activation via overexpression of the intracellular domain of Notch1 (NICD1) in Notch1-deficient MEFs. In contrast, constitutive activation of Notch1 signaling by introducing NICD1 into primary human dermal fibroblasts (FF2441), which caused pan-Notch activation, inhibited cell growth and motility, whereas cellular inhibition was relievable when the Notch activation was countered with dominant-negative mutant of Master-mind like 1 (DN-MAML-1). Functionally, "Notch-activated" stromal fibroblasts could inhibit tumor cell growth/invasion. Moreover, Notch activation induced expression of Wnt-induced secreted proteins-1 (WISP-1/CCN4) in FF2441 cells while deletion of Notch1 in MEFs resulted in an opposite effect. Notably, WISP-1 suppressed fibroblast proliferation, and was responsible for mediating Notch1's inhibitory effect since siRNA-mediated blockade of WISP-1 expression could relieve cell growth inhibition. Notch1-induced WISP-1 expression appeared to be Wnt11-dependent, but Wnt1-independent. Blockade of Wnt11 expression resulted in decreased WISP-1 expression and liberated Notch-induced cell growth inhibition. These findings indicated that inhibition of fibroblast proliferation by Notch pathway activation is mediated, at least in part, through regulating Wnt1-independent, but Wnt11-dependent WISP-1 expression.
|
[
{
"section_content": "Fibroblasts are key components of the interstitial tissue present in most organs of the body [1].They provide a delicately balanced tissue-specific ECM that partitions the interstitial space between tissue cells, blood vessels and nerves.Fibroblasts play an important role in not only supporting tissue architecture, but also participating in maintenance of tissue homeostasis.Fibroblasts generate soluble proteins including growth and differentiation factors [2] and remodelling enzymes, for example, matrix metalloproteases (MMPs) [3].These important cells are also involved in synthesis of ECM, such as collagen and fibronectin [4].Fibroblasts are known to play a role in a variety of fibrotic disorders (fibrosis/sclerosis).Most recently, these cells have gained increasing attention since they are important components of the supporting stroma in a variety of solid tumors.Tumors have been characterized as a type of ''wound that does not heal'' [5] and are now viewed as ''organs'' which have a unique microenvironment and specific stromal compartment.Tumor stroma is comprised of inflammatory cells, endothelial cells, fibroblasts and ECM.Fibroblasts in tumor tissues have been termed carcinoma-associated fibroblasts (CAFs), tumorassociated fibroblasts (TAFs) or cancer-associated fibroblasts (CAFs) (herein termed as cancer-associated fibroblasts (CAFs)) [6].CAFs are postulated to promote tumor growth through direct stimulation of tumor cell proliferation and promotion of tumor angiogenesis.Fibroblasts, thus, may represent a new therapeutic target for modulating stroma-associated tissue regeneration and tumor growth. In normal adult tissue, resident fibroblasts are maintained in a relatively quiescent state in which they are involved in slow turnover of the ECM.Fibroblasts, once activated, undergo a change in phenotype from the quiescent state to a proliferative and contractile phenotype termed myofibroblasts (sometimes termed ''activated fibroblasts'').Myofibroblasts actively produce growth factors and ECM, display an elongated spindle shape, and express contractile a-smooth muscle actin (a-SMA) and vimentin [7].Myofibroblasts can arise from the local, resident fibroblasts or from circulating mesenchymal precursors/stem cells [8], and even from epithelial cells via epithelial mesenchymal transition (EMT) [9]. The Notch signaling pathway is an evolutionarily conserved signaling cascade that regulates a variety of cellular activities including proliferation, differentiation, quiescence and death [10].The Notch receptor and its ligands are transmembrane proteins whose signaling requires cell to cell contact between neighboring cells.Mammals have four Notch receptors (Notch1-4) and five Notch ligands which fall into two classes: Delta-like (Dll) and Jagged.Activation of Notch receptors is triggered by interaction with Notch ligands on adjacent cells.The receptor-ligand binding results in proteolytic cleavage (by TACE and c-secretase) of NICD from the membrane bond Notch.NICD subsequently translocates into the nucleus where it binds to CSL (CBF1/Suppressor of Hairless/Lag-1)/RBP-Jk and recruits Mastermind-like (MAML) to form a ternary complex that functions as a transcriptional activator of Notch target genes.Notch target genes include those belonging to the Hes and Hey families [11].The diverse outcome of Notch activation is dependent on several factors including the specific timing, the signal strength/gene dosage, and the cell type and context [12][13][14]. The role of Notch signaling in fibroblasts is poorly studied.In this work we investigated the function of Notch signaling in regulating the cell growth of fibroblasts through in vitro loss-/gainof-function approaches.We observed a suppressive effect of activation of Notch signaling on fibroblast proliferation.We demonstrated that the inhibitory effect of Notch signaling is partially mediated by the induction of WISP-1 (CCN4) through a Wnt11-dependent mechanism in fibroblasts. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "To study the physiological function of Notch1 in regulating fibroblast proliferation and migration, we deleted Notch1 gene in MEFs isolated from Notch1 Flox/Flox mouse embryos at E13.5 by transducing cells with Cre/lentiviruses versus GFP/lentiviruses (control) in vitro.Greater than 95% cells were transduced based on the percentage of GFP-positive cells observed under fluorescence microscopy.A similar percentage of Cre-positive cells were achieved using same MOI of Cre/lentiviruses (data not shown).Knock-down of Notch1 was confirmed by Western blot analysis (Figure 1A) a week after transduction.A small residual Notch1 expression observed can be explained by non-100% transduction of MEFs by Cre/lentiviruses.Notch1-deficient (Notch1 2/2 ) MEFs displayed faster cell growth rate as measured by MTT assay compared to (Notch1 Flox/Flox ) MEFs (Figure 1B).Cell morphology of the Notch1 2/2 MEFs appeared to be unaltered compared to the Notch1 Flox/Flox MEFs.Cell motility was increased in Notch1deficient (Notch1 2/2 ) versus Notch1 Flox/Flox MEFs as demonstrated by recording the trafficking of 15 randomly selected MEFs for 20 hours in an in vitro cell scratch assay (Figure 1C).To functionally validate the phenotypes observed in Notch1-deficient (Notch1 2/2 ) MEFs, we transduced these cells with NICD (an active form of Notch1) and GFP (control), respectively, using lentiviral vectors to reconstitute Notch1 activation.It was observed that phenotypic changes resulted from Notch1 deletion were reversible by reconstitution of Notch1 activation (Figure 1D).Notably, constitutive activation of the Notch1 pathway by NICD overexpression even resulted in a significant growth inhibition of Notch1 null MEFs (Notch1 2/2 ) compared to wild type of MEFs (Notch1 +/+ ).This is consistent with what we observed in human dermal fibroblasts (FF2441) (see below).Overall, these results indicate that Notch1 signaling in MEFs exerts a suppressive effect on cell growth and motility. ",
"section_name": "Deletion of Notch1 Increases Cell Growth and Motility of MEFs",
"section_num": null
},
{
"section_content": "To further study the role of Notch signaling activation on cell growth of fibroblasts, we aimed to examine the effects of constitutive Notch1 activation on proliferation of primary human dermal fibroblasts (FF2441) [14].To this end, we transduced FF2441 cells with lentiviral vectors encoding either the GFP marker gene or the NICD1 gene linked to the GFP marker gene via internal ribosome entry site (IRES) to express GFP and NICD1 independently [15].The NICD1-GFP-transduced fibroblasts simultaneously expressed NICD1 and GFP.At an MOI of 5, greater than 95% of transduced fibroblasts expressed GFP as observed by fluorescence microscopy (data not shown).Expression of NICD1 in transduced FF2441 cells was confirmed by immunoblotting analysis (Figure 2A).Interestingly, full-length of Notch1 (,250 Kd) was undetectable in fibroblasts transduced with GFP/lenti, while detectable in fibroblasts transduced with NICD-GFP/lenti.It suggested that constitutive expression of exogenous NICD induced expression of endogenous Notch1.We further examined the gene profiles of Notch pathway components using PCR array, we observed a ''self-propagated'' pan-Notch activation mechanism induced by enforced expression of exogenous NICD1 in human fibroblasts.We found that constitutive expression of NICD1 resulted in the up-regulation of gene expression of multiple Notch pathway components in FF2441 cells, including ligands (Dll1, Jagged1), receptors (Notch1, 3, and 4), and target genes (Deltex1, Hes1, Herp2, and Hrt3).The changes in gene induction were showed in Figure 2B.Thus, hyper-activation of Notch1 pathway caused a pan-Notch signaling activation.These results thus revealed a ''self-propagated'' pan-Notch activation mechanism induced by enforced expression of exogenous NICD1 in human fibroblasts. ",
"section_name": "Constitutive Expression of Exogenous NICD1 Induces Pan-Notch Activation in Human Fibroblasts",
"section_num": null
},
{
"section_content": "We therefore investigated the effect of enforced activation of Notch1 pathway on cell biology of human fibroblasts.The constitutive activation of Notch1 by introduction of NICD into FF2441 cells significantly inhibited cell growth and motility in human dermal fibroblasts.As determined by MTT assay, cell proliferation rate of FF2441-NICD1-GFP significantly decreased compared to FF2441-GFP cells (Figure 3A).However, such a phenotypic change was reversible when the Notch activation was countered with DN-MAML-1, an antagonist of Notch pathway activation.As shown in Figure 3B, cell growth rate of FF2441-NICD1-GFP-DN-MAML-1 was significantly re-boosted compared to the control (FF2441-NICD1-GFP-Mock).Moreover, Notch pathway activation retarded the cell growth of FF2441, but did not induce cell apoptosis as no increase in apoptotic cells was detectable by TUNEL assay (Figure 3C).Constitutive Notch pathway activation down-regulated expression a panel of cell cycle genes, including cyclin D2, cyclin C, cycling E1 and cdk 4, 6, 7 and 8 while up-regulated expression of CDK inhibitor 3 (Table 1), suggesting that Notch pathway activation results in cell cycle arrest in human dermal fibroblasts.Similarly, as determined by time-lapse photography to track and measure cell trafficking in a modified scratch assay (in vitro wound healing assay), the average distance traveled by cells toward the middle line of the gap was smaller at all time points for FF2441-NICD1-GFP compared to FF2441-GFP cells.Velocity of FF2441-NICD1-GFP versus FF2441-GFP cells was 14.8+/24.9(mean +/2 SD) versus 35.9+/26.1 mm/hour (data not shown).These observations indicated that constitutive activation of Notch pathway inhibits cell proliferation and migration in human dermal fibroblasts.Taken together, the above experiments revealed that deletion of Notch1 increases cell growth and motility in MEFs, while activation of Notch pathway inhibits cell growth and motility in human dermal fibroblasts.These data consistently pointed to a role of Notch signaling in modulating the growth and motility of fibroblast cells. ",
"section_name": "Enforced Activation of Notch Pathway Inhibits Human Fibroblast Proliferation, but Did Not Induce Cell Apoptosis",
"section_num": null
},
{
"section_content": "To further study the correlation between the Notch pathway activation and the biological activity of fibroblasts, we examined the status of Notch signaling in cultured, proliferating FF2441, which were basically ''activated'' due to stimulation of serum and growth factors in the culture medium, versus quiescent FF2441 cells, which were induced by cell-contact inhibition and serum starvation.The expression of Notch receptors, ligands and known target genes (including Hes and Hey) were analyzed by the RTu Profiler TM PCR Array system.None of the Notch signaling pathway genes was significantly expressed in the proliferating fibroblasts.In drastic contrast, the expression of Dll-1, Jagged-2, Notch1, Notch-3, Notch-4, TACE (ADAM-17) and Hey-1 were significantly up-regulated (.1.5-fold) in quiescent fibroblasts (Table 2).The increased protein expression of Hey-1 (in quiescent compared to proliferating fibroblasts) was confirmed by immunoblotting analysis (Figure 4).These data indicate that the Notch pathway is maintained in a less-activated or inactivated status in proliferating human dermal fibroblasts whereas when these cells become quiescent, they upregulate several Notch signaling components.Thus, the status of Notch signaling appears to be tightly correlated with the biological activity of fibroblasts. ",
"section_name": "Status of Notch Signaling Is Correlated with the Biological Activity of Fibroblasts",
"section_num": null
},
{
"section_content": "To address the potential biological relevance of Notch pathway activation in fibroblasts, we tested the role of ''Notch-activated'' fibroblasts as stromal cells in modulating the growth and invasion/ migration of melanoma cells since it is well established that stromal fibroblasts/CAFs play a critical role in regulating tumor growth and metastasis.For this purpose, a 3D skin melanoma reconstruct model, which resembles human physiological condition, was utilized.Skin reconstructs consist of a 'dermis' of collagen with embedded fibroblasts (FF2441-NICD1-GFP versus FF2441-GFP) and an 'epidermis' of multi-layered keratinocytes with equal number of metastatic melanoma cells (1205Lu) [16] [17].After 14 days in culture, more 1205Lu cells were countable and many 1205Lu cells were able to invade into 'dermis' in which FF2441-GFP were embedded.Strikingly, invasion/migration of melanoma cells into 'dermis', in which FF2441-NICD1-GFP were embedded, was remarkably suppressed, and less 1205Lu cells were detectable (Figure 5).These data indicated that ''Notch-activated'' fibroblasts inhibited growth of melanoma cells in ''skin'' and their invasion into 'dermis'. ",
"section_name": "''Notch-Activated'' Stromal Fibroblasts Inhibited Tumor Growth/Invasion in a 3D Skin Melanoma Model",
"section_num": null
},
{
"section_content": "To understand the signaling mechanisms for the pronounced effects of the Notch signaling on cell biology of fibroblasts, we examined the gene expression profile of Notch pathway activation in FF2441 cells using quantitative Human Notch Signaling Pathway RT 2 Profiler TM PCR array, and found an approximately 6.5-fold increase in the gene expression of WISP-1/CCN4 in FF2441-NICD1-GFP compared to FF2441-GFP cells (Figure 6A).To validate the observed up-regulation of mRNA of WISP-1/ CCN4, we conducted immunoblotting analyses and confirmed an up-regulated protein expression of WISP-1/CCN4 in FF2441-NICD1-GFP compared to FF2441-GFP cells (Figure 6B).Consistently, levels of WISP-1/CCN4 protein were down-regulated in Notch1-deficient (Notch1 2/2 ) MEFs compared to Notch1 Flox/Flox MEFs as demonstrated by immunoblotting analyses (Figure 6C).Transcriptional activation of WISP-1 gene was also tested in FF2441-NICD1-GFP versus FF2441-GFP cells.Cells were transduced with WISP1 promoter-Luc2/Lentivirus, control RPL10PROM/Lentivirus and R01_PROM/Lentivirus, respectively, and lucifease activities were measured 48 hours posttransduction.As shown in Figure 6D, constitutive activation of Notch1 significantly increased WISP-1 promoter activity in fibroblasts compared with control.Thus, these experiments identified WISP-1/CCN4 as one of the down-stream targets of Notch pathway in fibroblasts.An in silico analysis of CSL binding site in the promoter of WISP-1 gene did not reveal a matching sequence, implicating that Notch indirectly regulates WISP-1 gene expression.This is consistent with our findings showed below that the regulation of WISP-1 by Notch is mediated by Wnt-11 in fibroblasts. ",
"section_name": "Effect of Notch Signaling on Regulating WISP-1/CCN4 Expression in Fibroblasts",
"section_num": null
},
{
"section_content": "Since constitutive activation of Notch1 pathway resulted in a cell growth inhibition in fibroblasts, we sought to investigate a potential role of WISP-1/CCN4 in mediating Notch-induced growth control of fibroblasts.To examine for a potential biological function of WISP-1/CCN4 in regulating cell growth in fibroblasts, we performed MTT assay to test the effect of recombinant human WISP-1/CCN4 on cell proliferation in FF2441 cells since WISP-1/CCN4 is a soluble factor secreted by cells.Addition of recombinant human WISP-1/CCN4 into the cell culture medium significantly inhibited the growth rate of FF2441 cells, but only in the presence of 10% FBS in the culture medium.In serum-free medium, no significant inhibition was achievable.Two dosages of WISP-1/CCN4 were tested, and both 10 ng/mL and 200 ng/mL achieved comparable inhibition efficacy (Figure 7A).Similarly, supplementation with recombinant human WISP-1/CCN4 significantly slowed cell growth rate of Cre/MEFs in the presence of 10% of FBS, but not in the absence of serum.Only the higher dose (200 ng/mL) of WISP-1/CCN4 was able to suppress cell growth in Cre/MEFs, (Figure 7B), suggesting variable sensitivity of different types of fibroblasts in responding to WISP-1/CCN4.The serum-dependent effect of WISP-1/CCN4 on fibroblast proliferation suggested that WISP-1/CCN4 interferes with growth factor(s) in FBS that induce proliferation.However, WISP-1 does not appear to induce a direct growth inhibition signal.In support of this concept, we determined that addition of recombinant WISP-1/CCN4 (200 ng/mL) inhibited serum-induced phosphorylation of Erk1/2 (the signaling cascade delivered from the MAPK pathway ultimately regulates the cell cycle machinery) in human fibroblasts (Figure 7C, 7D) which were starved overnight with serum-free medium and re-stimulated with FBS (10% in the culture medium) in the presence of WISP-1/CCN4.These results demonstrated a specific serum-dependent inhibitory effect of WISP-1/CCN4 on the cell growth of fibroblasts that has not been previously reported. ",
"section_name": "WISP-1/CCN4 Suppresses Fibroblast Proliferation",
"section_num": null
},
{
"section_content": "Based on the findings detailed above, we hypothesized that the inhibitory effect of Notch pathway activation on cell growth of fibroblasts may be mediated through up-regulating the production of WISP-1/CCN4 which, in turn, exerts its suppressive effect through either autocrine or paracrine mechanism.To test this hypothesis, we carried out siRNA-mediated gene silencing experiments.Specific siRNA targeting human WISP-1/CCN4 and scrambled control siRNA were introduced into FF2441-NICD1-GFP cells respectively by transient transfection.Knockdown of WISP-1/CCN4 expression was confirmed by immunoblotting analyses 48 hours after transfection (Figure 8A).The siRNAs-transfected FF2441-NICD1-GFP cells (wherin Notch is constitutively activated) were subjected to MTT assay to examine cell growth rate.We observed that specific interference with the WISP-1/CCN4 expression partially, yet significantly relieves the inhibitory effect of Notch activation on cell growth (Figure 8B).The cell growth rate of FF2441-NICD1-GFP cells transfected with specific WISP-1 siRNA, but not control siRNA, was partially restored, suggesting that WISP-1/CCN4 is one of the critical functional mediators of Notch signaling in regulating fibroblast proliferation.These data demonstrated that inhibitory effects of Notch signaling on human fibroblast proliferation are mediated in part by WISP-1/CCN4.That is, WISP-1/CCN4 was demonstrated to be one of functional down-stream targets of Notch signaling in fibroblasts. Notch Signaling-Induced WISP-1/CCN4 Expression Is Wnt1-Independent, but Wnt11-Dependent in Fibroblasts WISP-1/CCN4 was originally reported to be induced by Wnt1 [18].However, our PCR array studies did not show that the levels of Wnt1 gene in FF2441-NICD1-GFP are elevated compared to that in FF2441-GFP cells.Immunoblotting analysis confirmed the PCR array findings on Wnt1 expression (data not shown).Alternatively, we observed that the expression of Wnt11 gene was up-regulated .3-foldby Notch pathway activation (Figure 9A).Immunoblotting analyses confirmed these findings on the protein levels (Figure 9B).To explore whether Wnt11 is responsible for the induction of WISP-1/CCN4 in human fibroblasts, we blocked Wnt11 expression by siRNA approach and investigated whether inhibition of Wnt-11 expression results in the modulation of WISP-1/CCN4 expression in FF2441-NICD1-GFP cells.As shown in Figure 9C, the upregulation of WISP-1/ CCN4 was reversed in FF2441-NICD1-GFP cells when Wnt11 was inhibited by siRNA.As a consequence, cell growth in FF2441-NICD1-GFP cells was rescued (Figure 9D).These data strongly suggest that Notch signaling-induced WISP-1/CCN4 expression in human fibroblasts is mediated through Wnt11, but not Wnt1.Thus, these data identified a novel Wnt1-independent, but Wnt11dependent mechanism for the induction of WISP-1/CCN expression in fibroblasts and thereby, for the first time, established a linkage between Notch, Wnt11 and WISP-1/CCN4. ",
"section_name": "Inhibitory Effect of Notch Signaling on Human Fibroblast Proliferation Is Partially Mediated by WISP-1/CCN4",
"section_num": null
},
{
"section_content": "Cell cycle and cell growth of fibroblasts are tightly controlled.In normal uninjured tissues, resident fibroblasts are maintained in a relatively quiescent state.When tissue is injured or undergoing tumorigenesis, quiescent resident fibroblasts are stimulated by inflammatory factors or tumor cell-derived stimulatory factors, and activated.The involvement of Notch signaling in regulating cell growth of fibroblasts was previously mostly unexplored.A few prior studies provide inconsistent results with respect to Notch's role in regulating the fibroblast cell cycle.For example, a study showed that Notch1 activation promotes the G 1 -S transition of the cell cycle by inducing the expression of Skp2 and the consequent degradation of the CDK inhibitor p27 in 3T3 mouse fibroblasts [19].In contrast, activation of Notch signaling via either overexpression of NICD or stabilization of NICD by ablation of SEL-10 (Fbxw7), a negative regulator of Notch signaling, resulted in cell cycle arrest and apoptosis in mouse embryonic fibroblasts (MEFs) [20].The results of the latter study are consistent with our findings.In other prior work also consistent with our findings, it was reported that inhibition of Notch signaling by soluble forms of the Dll1 and Jagged1 ligands was able to induce fibroblast growth factor receptor (FGFR)-dependent transformation of NIH 3T3 fibroblasts in vitro [21].These studies and ours point to Notch signaling as a negative regulator or 'break' on fibroblast cell growth. An overall assessment of the prior literature and our novel findings appears to indicate that the status of Notch signaling is tightly correlated with the growth characteristics of fibroblasts.Deletion or inhibition of Notch1 signaling exempts fibroblasts from at least one growth control mechanism and cells proliferate faster as occurred in Notch1-deficient (Notch1 2/2 ) MEFs, whereas enforced activation of the Notch pathway inhibits cell growth of human dermal fibroblasts.It is, therefore, postulated that deregulation of Notch signaling may be involved in the pathophysiology of some disorders in which fibroblasts are involved.Our study employing Notch-engineered fibroblasts as stromal cells for modulating tumor cell growth/invasion provides an example to demonstrate the biological relevance of Notch activation in fibroblasts.It also suggests a paracrine effect of ''Notch-activated'' fibroblasts on other types of cells.In this regard, we have recently demonstrated that Notch-induced WISP-1 expression in fibroblasts is responsible for the inhibition of melanoma growth [22].Our work implies that Notch pathway is likely inactivated in CAFs of tumors, such as melanoma, since ''Notch-activated'' fibroblasts suppress melanoma cell growth/ invasion.This new concept has not been previously tested.Future studies will be required to determine the potential correlation between the status of the Notch activation and biological function of CAFs in tumors. Our work also revealed a previously unexplored concept.Constitutive activation of Notch1 pathway induces activation of pan-Notch pathways.It indicates existence of a mechanism for Notch signaling undergoing ''self propagation''.It is, however, unclear whether this is a unique phenomenon in fibroblasts, or it is a fairly universal mechanism for Notch signaling in other cell types.Although more studies are required, the latter appears to be the case, since we have found that overexpression of NICD1 in other cells, for example, human melanocytes, can induce the expression of several Notch ligands and receptors (ZL unpublished data).Ross et al. also observed that activation of Notch signaling induces Jagged-1 expression in C2C12 and NIH3T3 cells [23].However, unlike the concept of self-propagation of initial signal, as suggested by our data, it was speculated that the induced Jagged-1 has no apparent authorizing effects on Notch signaling but can promote signaling in naı ¨ve cells.That is, it was previously attributed to a mechanism through which Notch signaling can be relayed from cell to cell. A previously unknown signaling mechanism discovered by this work is the identification of WISP-1/CCN4 as one of functional mediators in delivery of the inhibitory effect of Notch signaling on fibroblast proliferation.The data not only locate WISP-1/CCN4 as a down-stream target gene of Notch signaling, but also unveil a new target for potential therapeutic manipulation.The molecular mechanism regarding how WISP-1/CCN4 exerts its serumdependent inhibitory effect on cell growth of fibroblasts remains an open question for future study.WISP-1 has been reported to function as a pro-mitogenic factor in mediating TNF-a-induced cardiac fibroblast proliferation [24].The reason as to why WISP-1 exerts paradoxical biological effects remains unknown.Presumably, it is determined by other cooperative signaling(s) induced by up-stream signal, because TNF-a stimulates cell growth of cardiac fibroblasts whereas Notch signaling suppresses fibroblast proliferation.Alternatively, it is simply cell type-dependent.It has been reported that WISP-1 binding to human skin fibroblasts is mediated through interaction with cell surface decorin and biglycan [25].It may prove worthwhile to investigate whether decorin and biglycan are responsible for mediating WISP-1's action on fibroblast growth inhibition.WISP-1 was initially identified as a Wnt1 responsive target [18], and belongs to the CCN family, which includes connective tissue growth factor (CTGF), cysteine-rich-61 (CYR61), and nephroblastoma overexpressed (NOV) [26].NOV (CCN3) has been shown to associate with Notch1 extracellular domain and exert a positive effect on Notch signaling in inhibiting myoblasts differentiation [27], while our findings reveal that WISP-1/ CCN4 functions as a down-stream target of Notch signaling in fibroblasts.These studies provide representative examples to demonstrate the interaction between Notch and CCN family.Until now, Wnt1 is the only member in the Wnt family known to induce WISP-1.Wnt4 is unable to up-regulate WISP-1 expression in the same experimental setting as Wnt1 does [18].The promoter of WISP-1 has been shown to be activated by both Wnt 1 and bcatenin expression.TCF/LEF sites played a minor role, whereas the CREB site played an important role in the transcriptional activation [28].Our observation that WISP-1 responds to Wnt11, but not Wnt1, mediates the observed effects, expands the scope of the Wnt family members involved in regulating WISP-1 and suggests that the Wnt/WISP axis may be cell type-specific.However, unlike Wnt1, which is known to activate the canonical Wnt/b-catenin pathway, Wnt11 is classified as a non-canonical signaling.Wnt11 is essential for the development of the heart and kidney [29][30][31][32], and is also implicated in cancer [33][34][35][36].Wnt11 signaling is thought to function in part by inhibiting the activity of the b-catenin-dependent Wnt pathway [37].We have not evaluated the activity of b-catenin in response to Wnt11 signaling in this study.Future work will be required to elucidate the Wnt11 down-stream pathway responsible for regulating WISP-1 expression in fibroblasts. In summary, we herein report an inhibitory role of Notch signaling in regulating cell growth of fibroblasts, which is partially mediated by the induction of WISP-1/CCN4 through a Wnt11dependent, but Wnt1-independent mechanism.Our study establishes a functional linkage between Notch, Wnt11 and WISP-1/ CCN4, and suggests a central role for Notch in coordination between these.The new findings lead us to postulate that Notch signaling in fibroblasts could potentially be implicated in some pathologic states featuring fibroblast growth deregulation. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Recombinant human WISP-1/CCN4 was purchased from R & D Systems (Minneapolis, MN).SDS-polyacrylamide gels were obtained from Invitrogen (Carlsbad, CA).X-ray films were purchased from Kodak (Rochester, NY).All other chemicals and solutions were from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. ",
"section_name": "Reagents",
"section_num": null
},
{
"section_content": "Notch1 Flox/Flox mice were established as described previously [38].Animal experiments were approved by the Institute Animal Care and Use Committee of the University of Miami (IACUC #10-228).Primary human dermal fibroblasts (FF2441) were initiated as explant cultures from trypsin-treated and epidermis- stripped neonatal foreskin [14] and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20 mM L-glutamine (Invitrogen), 8 mM HEPES and 10% FBS (Hyclone, Logan, UT).The MEFs were isolated from Notch1 Flox/Flox mice using the method described previously [39] and cultured in DMEM with glutamine, HEPES and 10% FBS.All experiments were performed with MEFs at passage 3-10.Human metastatic melanoma cell line, 1205Lu (ATCC, #CRL-2812), was cultured in 2% FBS-W489 medium as described [14].All cells were incubated at 37uC in 98% humidified air containing 5% CO 2 . ",
"section_name": "Mice and Cells",
"section_num": null
},
{
"section_content": "Methods for generation of GFP/lenti and NICD1-GFP/lenti were described previously [15].Cre/lenti was constructed by inserting Cre gene into pHX' lentiviral vector [15].Production of pseudotyped lentivirus was achieved by co-transfecting 293 T cells (ATCC # CRL-11268 TM ) with three plasmids as described [15].The lentiviruses collected 48 hours post-transfection displayed titers of around 10 7 transducing units/ml in NIH/3T3 cells (ATCC, #CRL-1658 TM ).Retroviral vector MAML305/pBabe (DN-MAML1 (Myc-tagged)) and empty pBabe vector (Mock) were described previously [40].To infect target cells by lentiviruses and retroviruses, cells were exposed six hours to virus with MOI (multiplicity of infection) 5 in the presence of 4 mg/ml polybrene.Cells were then washed, cultured with regular complete medium for two additional days, and analyzed for protein expression by Western blot or pooled for subsequent analysis as indicated in individual experiments. ",
"section_name": "Recombinant Lentiviruses and Retroviruses",
"section_num": null
},
{
"section_content": "The Human Notch Signaling Pathway RTu Profiler TM PCR Array (# PAHS-059, SABiosciences, Frederick, MD) and Human Cell Cycle RTu Profiler TM PCR Array (#PAHS-020) quantitatively profiles the expression of 84 genes involved in Notch signaling and 84 genes involved in cell cycle control, respectively.Total RNA was extracted from cells using TrizolH (Invitrogen) and cDNA was synthesized using RTu First Strand Kits (SABiosciences).PCR array was carried out according to the manufacturer's protocol.The threshold cycle (Ct) values were used to plot a standard curve.All samples were normalized to the relative levels of b-actin, and results are expressed as fluorescence intensity in relative levels. ",
"section_name": "PCR Array",
"section_num": null
},
{
"section_content": "Western blotting was performed as described [15].Membranes were probed with Abs to Notch1 (a rabbit polyclonal antiserum directly against residues 1759-2095) [15], cleaved/activated Notch1 (an antibody recognizing Val1744 (ab52301, Abcam, Cambridge, MA)), phospho-MAPK (#9106, Cell Signaling Technologies, Danvers, MA), p44/42 MAPK (#9102, Cell Signaling Technologies), WISP-1/CCN3 (H-57, sc-25441, Santa Cruz Biotechnologies, Santa Cruz, CA), Wnt-11 (sc-50360, Santa Cruz Biotechnologies), Hey-1 (GTX42614, GeneTex, Irvine, CA) or b-actin (AC-15, Abcam).Myc-tagged DN-MAML1 was detected on Western blot by 9-B11 Ab (Santa Cruz).This was followed by probing with HRP-conjugated second Ab (Jackson Immunoresearch, PA) and subjected to ECL (Amersham Biosciences, Piscataway, NJ).Membranes were stripped and re-blotted as required in the individual experiment.To quantify the bands in the blots, autophotographs of Western blots were scanned by computerized densitometry (Molecular Dynamics). ",
"section_name": "Immunoblotting",
"section_num": null
},
{
"section_content": "Plasmids containing WISP1 promoter-Luc2 fragment (WISP1 PROM_01, ID: S113793) and two control fragments (RPL10PROM_01, ID: S108908, and R01_PROM, ID: S190001) were purchased from SwitchGear Genomics (Menlo Park, CA).WISP1 PROM_01 was digested by SacI and SalI, and both RPL10PROM_01 and R01_PROM were digested by MulI and SalI, respectively.,2.1 kb WISP1 promoter-Luc2 and two control fragments were isolated, blunted and inserted into SmaI cut pHX' vector.Cells were transduced with WISP1 promoter-Luc2/ Lentivirus, control RPL10PROM/Lentivirus and R01_PROM/ Lentivirus, respectively.48 hours post-transduction, 2610 4 cells/ well in 96 well plates (triplicates) were measured for luciferase activity.Luciferase assays were performed using Steady-GloH Luciferase Assay kit (Promega, Madison, WI) according to the manufacturer's protocol.Luciferase activity measurement was corrected by subtraction of readings of controls. ",
"section_name": "Construction of WISP1 Promoter-Luc2/Lentiviral Vector and Luciferase Assay",
"section_num": null
},
{
"section_content": "Cell growth was measured by MTT assay.MTT cell proliferation kits were purchased from BioVision Technologies (Exton, PA).Cell proliferation was measured according to the manufacturer's protocol.2,000-5,000 cells/well were cultured in 96-well plate and cultured in DMEM with or without FBS as indicated in individual experiment.Samples were assayed in triplicate and experiments were repeated three times. ",
"section_name": "MTT Assay",
"section_num": null
},
{
"section_content": "Cell apoptosis was detected by TUNEL based ApopTagH kit (S7100) from Chemicon (Billerica, MA) according to the manufacturer's protocol.Briefly, cells fixed with 4% formaldehyde were pre-treated with 3% H 2 O 2 for 5 minutes at room temperature followed by washing with PBS.After incubation with equilibration buffer for 15 minutes at room temperature and TdT enzyme at 37uC for 60 minutes, cells were treated with stop/wash buffer for 10 minutes after being agitated for 15 seconds at room temperature, followed by washing with PBS.Cells were then incubated with anti-digoxigenin conjugate for 30 minutes at room temperature followed by washing with PBS.Apoptotic cells were stained with colorimetric substrates diaminobenzidine (DAB).Serum-starved cells (72 hours) were used as positive control. ",
"section_name": "Apoptosis Assay",
"section_num": null
},
{
"section_content": "To measure cell motility in vitro, MEF/Cre and MEF/GFP cells were cultured overnight to sub-confluence in 24-well plate.A gap was created by scratching cells with a standard tip.Velocity of fibroblast migration (mM per minute) was recorded using timelapse photography at 10-minute intervals, for 20 hours.Fifteen randomly selected individual cells per well were tracked, and data were analyzed by ImagePro 5.0 software (MediaCybernetics, Silver Spring, MD). In vitro Three-Dimensional (3D) Skin Melanoma Model 3D skin melanoma model was prepared as described [16,17].A total of 3 mL of fibroblasts (FF2441-NICD1-GFP versus FF2441-GFP, 7.5610 4 cells/mL) in a 4:1 mixture of bovine type I collagen (Organogenesis, Canton, MA): MatrigelH (BD Bioscience) was added to each insert of tissue culture trays (Organogenesis) and were allowed to constrict in DMEM with 10% FBS for 7 days at 37uC.For epidermal reconstruction, human keratinocytes, isolated from human epidermis of neonatal foreskins and cultured as described, [17,41] were mixed with human metastatic melanoma cells (1205Lu) at a ratio of 5:1 in epidermal growth medium composed of three parts DMEM and 1 part Ham's F-12 supplemented with 2.4 M CaCl 2 , 0.18 mM adenine, 4 mM glutamine, 10 mg/ml selenium, 10 mM ethanolamine, 0.1 mM O-phosphoryl ethanolamine, 10 mg/mL insulin, 10 mg/mL transferrin, 20 pM tri-iodothyronine, 0.5 mg/mL hydrocortisone and 4 pM progesterone.A total of 5610 6 cells were seeded on each contracted collagen gel.Cultures were kept submerged in medium containing 1 ng/mL EGF and 0.1% dialysed newborn calf serum for 2 days, then in 0.2 ng/mL EGF and 0.1% dialysed newborn calf serum for another 2 days, and then were raised to the airliquid interface via feeding from below with medium containing 2% dialysed newborn calf serum.After 14 days, skin reconstructs were fixed with 4% paraformaldehyde and were embedded in paraffin.Cell growth of melanoma cells was measured by counting melanoma cell number per low power field (LPF, X10).The invasive capacity of melanoma cells was determined by measuring the % of area of melanoma cells occupied in a given LPF based on morphological evaluation using H&E staining.Data are calculated based on that from 5 randomly selected fields/section and totally 10 sections per group. ",
"section_name": "In vitro Cell Motility Assay and Time-Lapse Photography",
"section_num": null
},
{
"section_content": "Short interfering RNA (siRNA) targeting the human form of WISP-1/CCN4 (sc-39335), Wnt-11 (sc-41120) and control, nontargeting siRNA (sc-37007), along with Transfection Reagent (sc-29528), siRNA Transfection Medium (sc-36868) and siRNA Dilution Buffer (sc-29527) were purchased from Santa Cruz Biotechnology.The experiments were performed according to the manufacturer's protocol. ",
"section_name": "siRNA Gene Silencing",
"section_num": null
},
{
"section_content": "All data is expressed as mean 6 SD.Statistical analysis was carried out using paired Student's t-test.Values considered statistically significant were P,0.05. ",
"section_name": "Statistical Analysis",
"section_num": null
}
] |
[
{
"section_content": "We thank Dr. M. Herlyn for FF2441 cells.We also thank Dr. A. J. Capobianco for the critical reading of this manuscript and Dr. George McNamara for assistance with the time-lapse photography and data analyses. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "This work was supported by grants from the National Institutes of Health (R01DK-071084 and R01GM081570).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": "Author Contributions",
"section_num": null
}
] |
10.3390/cells12232687
|
Characterization of Three Somatic Mutations in the 3′UTR of RRAS2 and Their Inverse Correlation with Lymphocytosis in Chronic Lymphocytic Leukemia
|
<jats:p>Chronic lymphocytic leukemia (CLL) is a hematologic malignancy characterized by progressive accumulation of a rare population of CD5+ B-lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. CLL exhibits remarkable clinical heterogeneity, with some patients presenting with indolent disease and others progressing rapidly to aggressive CLL. The significant heterogeneity of CLL underscores the importance of identifying novel prognostic markers. Recently, the RAS-related gene RRAS2 has emerged as both a driver oncogene and a potential marker for CLL progression, with higher RRAS2 expression associated with poorer disease prognosis. Although missense somatic mutations in the coding sequence of RRAS2 have not been described in CLL, this study reports the frequent detection of three somatic mutations in the 3′ untranslated region (3′UTR) affecting positions +26, +53, and +180 downstream of the stop codon in the mRNA. An inverse relationship was observed between these three somatic mutations and RRAS2 mRNA expression, which correlated with lower blood lymphocytosis. These findings highlight the importance of RRAS2 overexpression in CLL development and prognosis and point to somatic mutations in its 3′UTR as novel mechanistic clues. Our results may contribute to the development of targeted therapeutic strategies and improved risk stratification for CLL patients.</jats:p>
|
[
{
"section_content": "B-cell chronic lymphocytic leukemia (CLL) is a mature B-cell neoplasm characterized by the progressive accumulation of a rare population of mature B-lymphocytes (classified as CD19+CD5+) in the blood and lymphoid organs [1].It is the most prevalent type of leukemia in the Western world with an overall incidence rate of 4.2 per 100,000 [2,3].CLL presents a gender disparity of approximately 2:1, with a higher risk associated with the male faction (6.1 per 100,000 in males compared to 3.1 in females) [4,5].In the United States alone, approximately 18,740 new cases of CLL are diagnosed each year (American Cancer Society).This comprises roughly 25-30% of all newly diagnosed leukemia cases and accounts for about 1.1% of all new cancer diagnoses.CLL primarily affects older individuals, with the typical age of diagnosis hovering at approximately 70 years [2].The Cells 2023, 12, 2687 2 of 15 incidence of CLL rises notably after the age of 50 and continues to increase with advancing age [6]. While increasing age is one of the most significant risk factors for CLL, certain genetic abnormalities have been associated with an increased risk of developing the disease.Cytogenetic studies reveal recurrent chromosomal alterations, including deletions at loci 11q, 13q, 17p, and trisomy 12 [7].Moreover, CLL patients with an unmutated IGHV status have a less favorable prognosis.Unmutated IGHV is associated with a more rapid disease progression, a shorter time for treatment, and reduced overall survival rate.In these patients, the IGHV gene has not undergone somatic hypermutation, suggesting that the precursor of the leukemic cells is a pre-germinal center (GC) B-cell less terminally differentiated than that of CLL with mutated IGHV [7].Although CLL has been extensively studied, the molecular mechanisms underlying disease development are still to be fully elucidated.Genes deregulated and/or altered in CLL have been identified and singled out as possible driver genes.This includes genes such as TP53, ATM, MYD88, NRAS, and KRAS, which are involved in fundamental cellular processes such as DNA repair, cell-cycle control, and Notch, Wnt, and B-cell receptor (BCR) signaling [8]. RAS proteins are a family of small guanosine triphosphate hydrolases (GTPases) that include well-known oncogenic players such as aforementioned K-RAS and N-RAS, alongside H-RAS.R-RAS2 belongs to the RAS-related subfamily of RAS proteins, which share approximately 60% amino acid identity with classic RAS.Seemingly, R-RAS2 also shares associated proteins that regulate its activation-inactivation cycles with its classical counterparts including guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) [9,10].R-RAS2 has been identified as a key player in immune system homeostasis.It interacts with both the B-and T-cell receptors (BCR and TCR, respectively), facilitating the generation of tonic survival signals from these receptors [11].In B-cells, R-RAS2 has been shown to be crucial for the effective establishment of a proper GC reaction by regulating B-cell metabolism [12]. Although early studies from the 1990s show that oncogenic mutations in RRAS2 (G23V, Q72L) have higher transformation potential than analogous mutations in classical RAS genes (G12V, Q61L), RRAS2 is mainly found overexpressed in its wild-type form rather than mutated [9,10].It has been described that R-RAS2 protein is overexpressed in different types of human malignancies, including skin cancers [13], oral cancers [14], and esophageal tumors [15].We have recently demonstrated a driver role for RRAS2 bearing the activating mutation Q72L, which causes T-cell acute lymphocytic leukemia, a leukemia of immature B-cells, a Harderian gland adenoma, and an ovarian cystadenocarcinoma in 100% of mice, among other malignancies [16].However, those mice do not develop leukemias or lymphomas of mature T-cell or B-cell origin.In contrast, in another recent publication, we demonstrated that overexpression of wild-type human RRAS2 bearing no mutations causes the development of B-cell chronic lymphocytic leukemia in 100% of mice, thus demonstrating that wild-type RRAS2 is an oncogene driver if overexpressed [17].The driver role in mice was paralleled by the finding of wild-type RRAS2 mRNA overexpression in 82% of human CLL blood samples.Moreover, RRAS2 overexpression was found to be higher in patients with full-blown CLL than in patients with the pre-malignant stage monoclonal B-cell lymphocytosis (MBL).In addition, higher RRAS2 overexpression was correlated with higher lymphocytosis, a higher percentage of CD19+CD5+ malignant cells in the blood, and a reduced number of platelets, all data indicating that RRAS2 mRNA expression levels are associated with a more aggressive disease and worse prognosis. In addition to the association between CLL aggressiveness and RRAS2 expression in patients, we found another powerful correlation in human disease that sustains our hypothesis suggesting RRAS2 overexpression is one of the key driver oncogenes in CLL.We found the existence of a single nucleotide polymorphism (SNP) (rs8570) that catalogues the change of the canonical G-nucleotide at position +124 after the stop codon in the RRAS2 3 UTR for a C [17].The presence of a C-allele at the SNP position, whether in homozygosis or heterozygosis, was strongly linked to higher RRAS2 mRNA expression and various indicators of worse prognosis. In this paper, we present a comprehensive investigation into the mutations occurring within the 3 UTR of RRAS2, revealing a striking pattern of recurrent mutations at positions +26, +53, and +180 after the stop codon.We show how these somatic mutations, distinct from the SNP rs8570, associate with RRAS2 mRNA expression and markers of CLL aggressiveness. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "Human blood samples were collected from volunteer patients with chronic lymphocytic leukemia (CLL) at the Hematology Unit of Salamanca University Hospital.The collection was carried out following the provision of written informed consent, and the study was authorized under the number PI 2019 03217.All participants, including both patients and healthy volunteers, belong to the Caucasian ethnicity.Fresh human peripheral blood mononuclear cells (PBMCs) were obtained through density centrifugation using a Lymphoprep™ (StemCell Technologies, Vancouver, BC, Canada) gradient of whole blood.These PBMCs were utilized for flow cytometry, RT-qPCR analysis, and gDNA extraction. ",
"section_name": "Human Cells",
"section_num": "2.1."
},
{
"section_content": "Human single-cell suspensions were incubated for 15 min with Ghost Dye™ 540 (Tonbo) in PBS to label and discard dead cells from analysis.Cells were washed with PBS + 2% FBS and incubated with fluorescently labelled antibodies for 30 min at 4 • C after.Afterwards, cells were washed in PBS + 2% FBS and data were collected on a FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA) cytometer.A minimum of 50,000 and a maximum of 200,000 events was acquired in every measurement.Analyses were performed using FlowJo software (vX.0.7,BD Biosciences, Franklin Lakes, NJ, USA). ",
"section_name": "Flow Cytometry",
"section_num": "2.2."
},
{
"section_content": "A total of 10 6 cells were harvested per sample.Then, 500 µL of lysis buffer (Tris-HCl pH 8 50 mM, NaCl 200 mM, EDTA 10 mM, SDS 1% and fresh proteinase K 0.2 mg/mL) were added to each sample and incubated overnight (ON) at 55 • C. The next day, gDNA was purified using phenol chloroform and resuspended in 100 µL of 10 mM Tris-HCl pH 8.5 depending on the pellet size obtained after the last centrifugation step. ",
"section_name": "gDNA Extraction",
"section_num": "2.3."
},
{
"section_content": "A total of 10 6 cells per patient sample were used for RNA extraction from PBMCs of CLL patients.RNA was isolated using the RNAeasy Plus Mini Kit (QIAGEN Sciences, Germantown, MD, USA).cDNA was synthesized with SuperScript III (Thermo Fisher Scientific, Waltham, MA, USA) using Oligo-dT primers.cDNA was used as the template to sequence the 3 UTR region of RRAS2.Specific oligonucleotides were used to detect the presence of the canonical or alternative allele in each position (26, 53, 124, 180; Table 1) by quantitative real-time PCR (qPCR).All qPCR readings were performed in triplicate using 100 ng cDNA, SYBR Green PCR Master Mix, and gene-specific primers (Table 1) in an ABI 7300 Real Time PCR System.The oligonucleotides in Table 1 were designed as described in [18].As a loading reference, specific oligonucleotides aligning to constitutive RRAS2 exons 3 and 4 were used (forward: GCA GGA CAA GAA GAG TTT GGA; reverse: TCA TTG GGA ACT CAT CAC GA).The obtained cycle threshold (Ct) values were used to normalize RRAS2 3 UTR mRNA expression in each individual patient relative to their respective exons 3 and 4 expression.This approach accounted for variability in gene expression across samples, ensuring accurate comparisons.Table 1 provides a detailed overview of the specific oligonucleotides utilized in the qPCR analysis to characterize mutations within RRAS2 3 UTR.Notably, the nucleotide in blue type represents the mutation site in the somatic mutations 26, 53, and 180 as well as the SNP (rs8570) at 124.The red nucleotides differ from the native gene sequence to enhance the correct separation of the allele populations according to the results in [18]. ",
"section_name": "Sequencing Strategy for Patients' Samples",
"section_num": "2.4."
},
{
"section_content": "A set of primers that expand constitutive exons 3 and 4 was used to measure mRNA expression of RRAS2 in patient PBMCs (forward: GCA GGA CAA GAA GAG TTT GGA; reverse: TCA TTG GGA ACT CAT CAC GA).Obtained cycle threshold (Ct) values were used to calculate mRNA levels relative to 18S rRNA expression using the 2 (-∆∆Ct) method.Outliers for RRAS2 expression were identified using ROUT model at Q = 0.1% to remove definite outliers from analysis. ",
"section_name": "RRAS2 Expression Measurement",
"section_num": "2.5."
},
{
"section_content": "Fluorescent dual-labelled probe technology, pre-developed by Applied Biosystems (Foster City, CA, USA) and custom made to target rs8570 SNP was used to sequence our CLL and healthy patients.A detailed description of the methodology employed by the custom-made TaqMan technology is available at [19].All qPCR reactions were performed using an initial cycle of 10 min at 95 • C for polymerase activation, followed by 40 cycles of 15s at 95 • C and 1 min at 60 • C. rs8570 allele distribution was assessed by plotting the relative fluorescent units (RFU) of both fluorophores in a scatter-plot representation. ",
"section_name": "Fluorescent Probes Method to Sequence rs8570 SNP in CLL Patients' Samples",
"section_num": "2.6."
},
{
"section_content": "",
"section_name": "Statistical Analysis",
"section_num": "2.7."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "We have previously shown that the SNP rs8570 in the 3 UTR of human RRAS2 is associated with higher mRNA abundance and a worse prognosis in CLL patients.Expression of the non-canonical C-allele in one or two dosages in leukemic cells was linked to higher RRAS2 expression than in leukemic cells carrying two copies of the canonical G-allele (GG) [17].Furthermore, the frequency distribution of G-and C-alleles at SNP rs8570 was not in Hardy-Weinberg equilibrium [20], suggesting the presence of selective pressure favoring the C-allele within the human CLL patient population.Recently, we developed a new quantitative PCR method to enhance the genotype characterization at the SNP position [19].Using this new method, we analyzed the frequencies of GG, GC, and CC genotypes at the SNP position in the blood of an extended cohort of CLL patients (n = 203) and compared these frequencies with those of a cohort of healthy blood donors (n = 235) (Figure 1A,B; all sample data is shown in Table S1).Frequency analysis confirmed that the SNP alleles were not in equilibrium in the CLL sample population (Figure 1A), whereas the two alleles were in equilibrium in the healthy donor population (Figure 1B).Contingency tests of GG, GC, and CC frequencies in the patient and healthy blood cohorts showed that the imbalance in allele distribution was caused by an increased frequency of CC homozygotes in the patient population at the expense of GC heterozygotes (Figure 1C).These results confirmed the idea that the C-allele at SNP rs8570 position in the 3 UTR of RRAS2 mRNA is enriched in homozygosity in leukemic cells.Since the C-allele is associated with higher mRNA abundance [17], these data further support the idea that overexpression of wild-type RRAS2 is behind the development of CLL. In contrast to KRAS, the presence of somatic mutations in the coding sequence of RRAS2 is very rare in human cancers.In CLL, a total of 1308 patient samples from four different studies showed no mutations in the coding sequence of RRAS2 (cBioportal.org).To further characterize the RRAS2 mRNA, we sequenced the 3 UTR region using the Sanger method.We uncovered additional mutations, alongside that of SNP rs8570, including mutations at positions +26 (G>T), position +53 (T>C), and position +180 (T>C) after the stop codon (Figure 2A).Since none of these alterations have been reported as SNPs in databases, we hypothesize that they arise from somatic mutations in CLL patients.Next, we designed two sets of primer pairs per position to evaluate the frequencies of the canonical and alternate alleles by RT-qPCR (see Section 2).The results of both PCRs were normalized by obtaining the ratio of the alternate allele to the canonical allele.Such a ratio was calculated for each of the collected CLL blood samples and plotted (Figure 2B-D).The median and mean of the alternate/canonical ratios were calculated for each position, and samples with ratios above the median were considered to express the somatic mutation at least in heterozygosity.Interestingly, some blood samples contained somatic mutations in two of the three positions and some in all three positions simultaneously (red dots in Figure 2B-D).Our data showed that 51% of the CLL samples contained at least one mutation at either one of the three positions (Figure 2E).Each of the three positions was mutated with similar frequency (37-38%), whereas 20% of the samples carried two mutations, with the double mutation +26+180 being the most common, and approximately 3% carried mutations at all three positions (Figure 2E).These results show that, in contrast to missense mutations in the RRAS2 gene, mutations in the 3 UTR at three specific positions are common in CLL. gency tests of GG, GC, and CC frequencies in the patient and healthy blood cohorts showed that the imbalance in allele distribution was caused by an increased frequency of CC homozygotes in the patient population at the expense of GC heterozygotes (Figure 1C).These results confirmed the idea that the C-allele at SNP rs8570 position in the 3′UTR of RRAS2 mRNA is enriched in homozygosity in leukemic cells.Since the C-allele is associated with higher mRNA abundance [17], these data further support the idea that overexpression of wild-type RRAS2 is behind the development of CLL. ",
"section_name": "Presence of Somatic Mutations in the 3 UTR of RRAS2 in CLL Patients",
"section_num": "3.1."
},
{
"section_content": "Next, we investigated if there was an association between the presence of mutations at positions +26, +53, and +180 and the allele distribution at the SNP rs8570 position.To this end, we first plotted the alternate/canonical ratios for each position in the CLL cohort classified as GG, GC, and CC SNP genotypes.We found that the ratios in favor of the alternate alleles were higher in samples with GC genotype than in GG and CC homozygotes, although they reached statistical significance only for position +26 when comparing GC and GG genotypes (Figure 3A).Calculation of the average number of mutations at any of the three positions in the 3 UTR showed a slightly higher incidence in GC heterozygotes than in GG homozygotes and clearly higher than in CC homozygotes (Figure 3B).Alternatively, we analyzed the results according to the frequency of GG, GC, and CC genotypes in the groups of patient samples classified by the presence of mutations at any of the three sites.Compared to the samples without mutation in the 3 UTR, the samples with mutation at any of the three sites showed a higher frequency of GC heterozygotes to the detriment of GG and CC homozygotes.Such enrichment in GC genotypes reached statistical significance for position +26 (Figure 3C).An analysis of allele frequencies for SNP rs8570 showed that such frequencies were not in Hardy-Weinberg equilibrium in both the group of samples with no mutations and the group of samples with mutations at position +26 (Figure 3D,E); the others were in equilibrium (not shown).The disequilibrium was due to enrichment for CC homozygotes in the unmutated group and enrichment for GC heterozygotes in the +26 mutation group. The 3 UTR of mRNAs has a general function in regulating mRNA stability, nuclear export, and translation efficiency [21].Since allelic composition at the rs8570 SNP position influences RRAS2 mRNA abundance [17], we investigated whether the somatic mutations at any of the three positions found here influence mRNA expression.First, we found that the group of patient samples with no mutations had significantly higher mRNA expression than the group of patient samples with at least one mutation (Figure 3F).The site-specific analysis showed that a mutation at any of the three positions resulted in significantly lower mRNA abundance than the no mutation group (Figure 3G).These results show that the presence of mutations at any of the three positions in the 3 UTR inversely correlates with RRAS2 mRNA expression.(D,E) Pie chart representation of the observed distribution of the GG, GC, and CC genotypes at the rs8570 SNP in blood samples of the cohorts of CLL patients bearing no mutations in the 3 UTR and bearing mutation at the +26 position.The Chi-squared value (χ 2 ) is greater than 3.84 for both groups such that the hypothesis that genotypes are at Hardy-Weinberg equilibrium is rejected with p < 0.025.(F) Violin plot showing all experimental points, the median, and the upper and lower quartiles for RRAS2 expression in all CLL patients in our cohort, classified according to the presence or not of any mutation in the 3 UTR.Significance was assessed using a non-parametric unpaired Mann-Whitney rank test (****, p < 0.0001).(G) Violin plot showing all experimental points, the median and the upper and lower quartiles for RRAS2 expression in all CLL patients in our cohort classified according to the presence or not of mutations at the indicated positions of the 3 UTR.Significance was assessed using a non-parametric unpaired Dunn's multiple comparisons test (****, p < 0.0001; ***, p = 0.0002; **, p = 0.0016).(H) Violin plot showing all experimental points, the median, and the upper and lower quartiles for RRAS2 expression in all CLL patients in our cohort, classified according to the SNP rs8570 genotype and to the presence or not of mutations at the three positions of the 3 UTR.Significance was assessed using a non-parametric unpaired Mann-Whitney rank test (**, p = 0.0067; *, p = 0.017).ns, not significant (p > 0.05). ",
"section_name": "Association of the Three 3 UTR Mutations with RRAS2 mRNA Abundance",
"section_num": "3.2."
},
{
"section_content": "Overexpression of RRAS2 mRNA is associated with a higher proportion of CLL cases at diagnosis versus pre-malignant monoclonal B-cell lymphocytosis (MBL), a higher proportion of leukemias bearing unmutated IgH gene, male sex, higher age, higher proportion of chromosomal anomalies, higher lymphocytosis, and lower platelet counts-all markers of poorer prognosis [22].Therefore, we next determined if there was any association between the three somatic mutations in the 3 UTR of RRAS2 and clinical data.We found that unlike for SNP rs8570, there was no significant difference in the percentage of patients with MBL versus full-blown CLL at diagnosis between unmutated and mutated samples at any of the three positions (Figure 4A).Likewise, we did not find significant differences in the distribution of mutated and unmutated samples according to the mutated/non-mutated IgH status (Figure 4B) or the existence of chromosomal aberrations by FISH (Figure 4C).There was slightly less male/female disequilibrium in patients with mutated versus unmutated 3 UTR, but it did not reach statistical significance (Figure 4D).Also, and unlike for SNP rs8570, there were not significant differences in age distribution (Figure 4E) or platelet counts (Figure 4F).Interestingly, we did find an inverse relationship between the presence of somatic mutations at any of the three positions in the 3 UTR and total leukocyte counts (Figure 4G), total lymphocyte counts (Figure 4H), and the percentage of CD19+CD5+ leukemic cells in blood (Figure 4I), compared to the samples with no mutations.These results suggest that malignant leukemic cell expansion in CLL patients is hampered by the acquisition of somatic mutations at any of the three sites of the 3 UTR of RRAS2. ",
"section_name": "Association of the Three 3 UTR Mutations with Clinical Traits",
"section_num": "3.3."
},
{
"section_content": "In this study, we describe three somatic mutations in the 3 UTR of RRAS2 mRNA in CLL patients.In contrast to the rarity of missense mutations in the RRAS2 gene, the 3 UTR harbors mutational hotspots at specific positions: +26 (G>T), +53 (T>C), and +180 (T>C) after the stop codon.Mutations within the 3 UTR of genes have historically been overshadowed by their counterparts within coding regions or canonical splice sites.However, the importance of these non-coding mutations is becoming increasingly evident, as they can disrupt RNA-protein interactions, microRNA binding sites, or other regulatory elements, thereby impacting gene expression and cellular phenotypes [23]. Our results reveal an intriguing relationship between RRAS2 mRNA expression and the three 3 UTR somatic mutations.The newly identified 3 UTR mutations present an inverse relationship with RRAS2 mRNA expression levels and correlate with more positive prognostic or less severe clinical factors (reduced lymphocytosis and improved platelet count).The reduction in RRAS2 mRNA expression levels could be behind the reduced lymphocytosis observed in CLL patients with these mutations.Thus, the effect of the three somatic mutations at the 3 UTR seems to exert the opposite effect to the expression of the alternate C-allele at the SNP rs8570 [17].Overall, our findings hint at a complex relationship between RRAS2 mRNA expression, the rs8570 SNP, and the three somatic mutations identified in the gene's 3 UTR.Higher RRAS2 expression is linked to the dose of the C-allele: CC homozygous leukemias express more mRNA than GC heterozygous, and those more than GG homozygous.Comparing RRAS2 mRNA expression between mutated and unmutated samples at any of the three 3 UTR positions for the three SNP genotypes, we show that mutation reduces RRAS2 expression, but the reduction is especially significant for GC SNP heterozygotes.In terms of frequency, the presence of either one of the three somatic mutations or their combinations is also associated with GC heterozygosity at the SNP position.It can be hypothesized that the three somatic mutations in the 3 UTR are selected to limit the effect of the SNP C-allele on mRNA expression, and that such effect is in cis.This could be the reason that the mutations are preferentially selected in rs8570 heterozygote (GC) patients because they have just one chromosome to be modified.In fact, after sequencing the entire 3 UTR region expanding the first 200 nucleotides after the stop codon, we find a preferential enrichment in the three mutations in the chromosome bearing the C-allele compared to the one bearing the G-allele.This conclusion is preliminary, given the relatively small number of GC heterozygotes at the SNP position that we have been able to sequence.If confirmed, the association in cis could indicate that mutations in positions +26, +53, and +180 could interfere with microRNAs or other regulatory mechanisms associated with the SNP in position +124. Our leading hypothesis consists of a compensatory mechanism at play.The 124 Callele (rs8570) has been associated with higher RRAS2 expression and, consequently, a worse disease prognosis [17].We propose that the presence of three newly identified 3 UTR mutations compensates for the negative attributes of the 124 C-allele and could potentially reduce the adverse effects of highly elevated RRAS2 expression.In addition to describing three somatic mutations in the 3 UTR sequence of the RRAS2 gene, we reinforce the idea that the SNP rs8570, and overall RRAS2 overexpression in its wild-type form, is linked to CLL development.The major finding, in addition to linking the alternate C-allele to higher mRNA expression, was that the distribution of GG, GC, and CC genotypes was tilted towards a higher abundance of CC homozygotes in the CLL patient cohort [17].Here, we have increased the number of patient samples in the CLL cohort and compared it with a cohort of healthy blood donors.Thus, we confirm the disproportion in favor of CC homozygotes within the CLL cohort and show that such disproportion is not found in the healthy population cohort, which is in Hardy-Weinberg equilibrium.However, we still do not know if the increased frequency of CC homozygotes within the cohort of CLL leukemic cells is originated from somatic G>C mutation at the SNP position or if it is due to a higher propensity of the human population with CC homozygosity at the SNP position to develop CLL. In the mouse model of RRAS2 overexpression driving CLL development, we found that with time, there is a selection in favor of leukemic cells with even higher expression of RRAS2 mRNA [17].Therefore, a higher proportion of leukemias bearing the SNP rs8570 in CC homozygosity is understandable due to its association with higher expression.By contrast, what could be the reason for the emergence of the three somatic mutations described here that result in lower RRAS2 expression and lower lymphocytosis?Our running hypothesis is that during the biological evolution of the leukemic CLL clones, excessive RRAS2 overexpression could lead to senescence or to a dead-end differentiation of the pre-malignant B-cell clones.Overall, the results shown in this paper further link wild-type RRAS2 overexpression to CLL development and clinical progression. ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "We have identified three mutational hotspots in the 3 UTR of the RRAS2 mRNA, affecting positions +25, +53, and +180 after the STOP codon, in 51% of our cohort of CLL patients.The presence of these three mutations alone or in combination results in reduced expression of RRAS2 mRNA in CLL samples compared to those not bearing mutations.The presence of any of the three mutations inversely correlates with the number of leukocytes, total lymphocytes, and leukemic cells in the blood.The association of these three mutations with reduced RRAS2 mRNA expression and diminished lymphocytosis reinforces the idea that RRAS2 overexpression is implicated in CLL development and disease progression. ",
"section_name": "Conclusions",
"section_num": "5."
}
] |
[
{
"section_content": "Funding: This work was supported by grants from the Spanish Association against Cancer (GC16173472GARC), Grant PID2019-104935RB-I00 from the 'Minsterio de Ciencia y Tecnología', Grant PID2022-136745OB-I00 funded by AEI/10.13039/501100011033 and by the \"European Union NextGenerationEU/PRTR\"; Grant P2022/BMD7209 (INTEGRAMUNE-CM) from the 'Comunidad de Madrid', the 'Fundación Ramón Areces', Instituto de Salud Carlos III (ISCIII: CIBERONCgroups CB16/12/00233, CB16/12/00351), the Health Council of the Junta de Castilla y León (GRS 2036/A/19), and Gilead (GLD15/00348).The publication is part of the project PDC2021-121170-I00 \"Leukomodel\", funded by the Spanish State Research Agency (AEI/10.13039/501100011033)and by the European Union under the \"NextGenerationEU\" program/PRTR. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells12232687/s1.Table S1.Clinical and genotypic data from CLL patients and healthy volunteers. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The data presented in this study are available upon request to the corresponding author. ",
"section_name": "Data Availability Statement:",
"section_num": null
},
{
"section_content": "Institutional Review Board Statement: All animal experiments were carried out at the facilities of the Centro de Biología Molecular Severo Ochoa in accordance with national and European guidelines.All the procedures were approved by the ethical committee of the Consejo Superior de Investigaciones Científicas (approval number: 197/2022).Blood samples from volunteer CLL patients were obtained from the Hematology Unit of the Salamanca University Hospital after providing written informed consent. Informed Consent Statement: Written informed consent was obtained from all patients. The authors declare no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "Institutional Review Board Statement: All animal experiments were carried out at the facilities of the Centro de Biología Molecular Severo Ochoa in accordance with national and European guidelines.All the procedures were approved by the ethical committee of the Consejo Superior de Investigaciones Científicas (approval number: 197/2022).Blood samples from volunteer CLL patients were obtained from the Hematology Unit of the Salamanca University Hospital after providing written informed consent. Informed Consent Statement: Written informed consent was obtained from all patients. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors declare no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.3389/fimmu.2019.00162
|
Adenosine A2A Receptor Stimulation Inhibits TCR-Induced Notch1 Activation in CD8+T-Cells
|
Notch receptors signaling is required for optimal T-cell activation and function. T-cell receptor (TCR) engagement can activate Notch receptors in T-cells in a ligand-independent fashion. In this study, we examined the role of adenosine A2A receptor (A2AR) signaling pathway in regulating the activity of Notch1 induced by TCR stimulation in CD8+T-cells. A selective A2AR agonist decreased Notch1 protein expression and Notch1 cleavage, and reduced transcripts of Notch1-target genes Hes1 and Myc in activated CD8+T-cells. Inhibition of TCR-induced Notch1 expression by an A2AR agonist was accompanied by increased cAMP concentration and mimicked by forskolin. This effect was associated with reduced IFN-γ and granzyme B production. The effect of an A2AR agonist was abrogated by a selective A2AR antagonist and absent in CD8+T-cells harvested from A2AR-/- mice. Stimulation of A2AR reduced Notch1 receptor levels by inhibiting upstream TCR signals, including ZAP70 phosphorylation, in turn impairing the generation of the active Notch1 intracellular domain (N1ICD). Direct activation of PKC with PMA and ionomycin bypassed A2AR-induced Notch1 inhibition. Overexpression of N1ICD in CD8+T-cells prevented the suppressive effects of an A2AR agonist on proliferation and cytokine release during activation. Our results identify the A2AR signaling pathway as an important regulator of TCR-induced Notch1 receptor activation in CD8+T-cells, and Notch as an important target of the immune suppressive effects of A2AR. We propose a mechanism whereby A2AR impairs CD8 T-cells function through inhibition of Notch1 receptor activation.
|
[
{
"section_content": "Notch signaling plays a pivotal role in the differentiation and function of several T-cell subsets [reviewed in (1)(2)(3)(4)].Notch proteins (Notch 1-4) are heterodimeric transmembrane receptors that are activated by engagement of transmembrane ligands (Delta-like 1, 3, 4, and Jagged 1, 2, although Delta-like 3 is likely an inhibitory ligand) (5).The interaction of Notch receptors with their ligands leads to subunit separation, followed by a trans-endocytosis of the Notch extracellular domain.This facilitates the cleavage of the Notch transmembrane subunit by metalloproteases of the ADAM family, which is followed by a second cleavage within the transmembrane domain by the γ-secretase complex.This generates the active intracellular domain of Notch (NICD) (1,2,6).NICD translocates into the nucleus where it associates with the transcriptional repressor CSL (CBF1-Suppressor of Hairless-LAG1), recruiting a co-activator complex, to modulate the transcription of several genes (7).In T-cells, ligand-independent activation of Notch1 receptor can be triggered through T-cell receptor (TCR) signals (8)(9)(10)(11), through mechanisms that require receptor endocytosis.Notch1 can upregulate its own expression in T-cells (12).TCR-induced up-regulation of Notch1 expression in peripheral T-cells is associated with increased proliferation and cytokine production, including IFN-γ (8), or the expression of the transcriptional regulator eomesodermin (EOMES), which in turn regulates the expression of CD8 effectors perforin and granzyme B (10).Transgenic expression of Notch1 renders CD8 T-cells resistant to tumor-induced immune suppression (13).Pharmacological inhibitors of γ-secretase, which prevent the generation of NICD (14,15), reduce proliferation and cytokine release from TCR-activated T-cells in a concentrationdependent manner (8,10).Notch2 also contributes to the activity of cytotoxic T-cells, controlling directly the transcription of granzyme B, independently of EOMES (16).The expression of Notch2 can be also up-regulated during T-cell activation and can, redundantly with Notch1, modulate proliferation and IFN-γ production in CD8+T-cells (13). Adenosine is an ATP metabolite that increases in the extracellular space in response to hypoxia and tissue injury, acting as an anti-inflammatory mediator that limits inflammation-induced damage (17).Adenosine can exert profound immunosuppression by acting on both lymphoid (18) and myeloid cells (19).The A2A receptor (A2AR) belongs to the family of adenosine receptors and is the predominant adenosine receptor type expressed in T-cells (20,21).This is a high-affinity, Gs-coupled receptor that upon activation increases cyclic AMP (cAMP), which in turn activates protein kinase A (PKA).Stimulation of A2AR suppresses TCR signaling in a cAMPdependent manner (22)(23)(24)(25)(26). Inhibition of TCR signaling by A2AR agonists is associated with reduced cytokine production, including interleukin-2 (IL-2) and interferon-γ (IFN-γ), and decreased cytotoxicity and proliferation (22)(23)(24)(25)(26)(27).Selective A2AR agonists may be used for the treatment of inflammatory diseases (28).Conversely, because A2AR in tumors contributes to induce profound immunosuppression, A2AR antagonists are considered potential novel cancer immunotherapeutics (29). Given the crucial role of A2AR signaling in limiting CD8+ Tcell responses, we evaluated its effects on Notch1 expression and signaling.We demonstrate that A2AR stimulation inhibits TCRinduced Notch1 expression and cleavage in activated CD8+Tcells, describing a novel mechanism through which adenosinergic molecules can suppress CD8+T-cells functions via A2AR. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "Splenocytes were collected aseptically from the spleens of naïve C57BL/6 mice (6-10 weeks old) (Charles River, Lecco, Italy) or from A2AR gene-deficient (A2AR-/-) C57BL/6-background mice (6-10 weeks old), kindly provided by M. Sitkovsky (Northeastern University).CD8+ T-cells were enriched with a negative selection kit (StemCell Technologies), according to manufacturer's instructions.Experiments were also performed in CD8+T-cells conditionally expressing transgenic Notch1 intracellular domain (N1IC CD8+T-cells) or floxed control cells (N1IC f/f CD8+T-cells) isolated, respectively, from previously described N1IC mice and N1IC f/f mice (13).Isolated cells, cultured in RPMI 1640 medium supplemented with 10% FBS, 4 mM L-Glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin and 50 µM 2-mercaptoethanol, were activated in plates coated with anti-mouse CD3ε (1 µg/ml; clone 145-2C11) and antimouse CD28 (0.5 µg/ml; clone 37.51) antibodies up to 72 h or left unstimulated (NS).In specific sets of experiments, CD8+ T-cells were activated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 200 ng/ml ionomycin (both from Sigma-Aldrich) or left unstimulated (NS). ",
"section_name": "CD8+ Cells Isolation and Activation",
"section_num": null
},
{
"section_content": "Cells were incubated with 1 µM A2AR agonist CGS-21680 (Tocris), 1 µM A 2A R antagonist ZM-241385 (Tocris), 1 µM γsecretase inhibitor PF-3084014 (SelleckChem) or with 10 µM adenylate cyclase stimulator Forskolin (Sigma-Aldrich), alone or in combination, 15 min before activation with anti-CD3ε/CD28 antibodies or PMA and ionomycin.PF-3084014 was dissolved in DMSO, whilst the other agents were solubilized in PBS (vehicle).Control cells (Ctr) received vehicle before activation as described above.In specific experiments, cells were incubated with the above agents 24 h after CD3ε/CD28-stimulation. ",
"section_name": "Cell Treatments",
"section_num": null
},
{
"section_content": "To measure proliferation, 5 × 10 5 isolated CD8+ T-cells were labeled with 1 µM carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, Life Technologies) for 10 min at 37 • C. Cells were then washed to remove excess CFSE and then treated with 1 µM CGS-21680, 1 µM PF-3084014, or 10 µM Forskolin before adding anti-CD3ε and anti-CD28 antibodies for 72 h.Proliferation was assessed by measuring CFSE fluorescence by flow cytometric analysis on a FACSCalibur cytometer (BD Biosciences).Data were analyzed using CellQuest software. ",
"section_name": "Proliferation Assays",
"section_num": null
},
{
"section_content": "Granzyme B and IFN-γ concentrations were measured in CD8+ T-cells supernatants after 72-h treatments as described above, by using specific ELISA kits (Thermo Fisher Scientific) according to the manufacturer's instructions. ",
"section_name": "ELISA Assays",
"section_num": null
},
{
"section_content": "1 × 10 6 cells treated as described above were collected, washed in ice-cold PBS and lysed in RIPA buffer to which were freshly added 1 mM Protease Inhibitor Cocktail (Thermo Scientific), 1 mM NaF and 1 mM sodium orthovanadate (all the reagents used for the lysis buffer were EDTA-free).Equal amounts of proteins were electrophoretically separated in SDS-Page gels and then transferred to PVDF membranes (Immobilion-FL Transfer Membrane).Bovine serum albumin (5% BSA, Sigma) was used to block non-specific binding sites.The following primary antibodies were used: anti-Notch1 (which specifically detects the Notch1 intracellular domain N1ICD, Val1744; D3B8), anti-Notch1 TM , (to detect the transmembrane Notch1 domain, D1E11) (both from Cell Signaling), anti-pZAP70 (pY319.17A)or anti-GAPDH, anti-β-tubulin or antiactin as controls (Santa Cruz Biothecnology).After incubation with appropriate secondary antibodies, immune-reactive protein bands were detected by enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, UK) and analyzed by Las4000 (GE Healthcare Life Sciences).The optical density of protein bands detected by Western blotting was calculated with ImageQuantTL (GE Healthcare). ",
"section_name": "Western Blot Analysis",
"section_num": null
},
{
"section_content": "Total RNA was isolated from treated cells using an RNeasy Mini kit (Qiagen) following the manufacturer's instructions.Reverse transcription was performed using the First strand cDNA synthesis Kit (Fermentase Life Science Co.).cDNAs were amplified by real-time PCR using iTaqTM SYBR Green Supermix with ROX (Bio-RAd).Primer sequences were: C for 1 min, followed by dissociation step.Granzyme B RT-PCR analysis was performed by using Taqman primers Mm00442837_m1 and GAPDH: Mm99999915_g1 as housekeeping gene (Applied Biosystems).Results were analyzed using the 7,300 system Software (Applied BioSystems). cAMP Assays cAMP levels were measured with the cAMP assay kit (Abcam), according to the manufacturer's instructions.cAMP levels were determined in 1 × 10 6 CD3ε/CD28-stimulated CD8+ T-cells pre-incubated with 1 µM CGS-21680 or 10 µM Forskolin.10 min later, cells were incubated with 0.1 M HCl at room temperature for 20 min.After gentle pipetting, samples were centrifuged for 10 min and supernatants collected.Samples were then assayed for cAMP levels. ",
"section_name": "Quantitative Real-Time RT-PCR",
"section_num": null
},
{
"section_content": "Unless otherwise stated, results are expressed as means ± SD.Data were analyzed with GraphPad Prism 6 (GraphPad Software).Two-tailed Student's t-test (2-group comparisons) or ANOVA (>2-group comparisons) were performed as appropriated, with Bonferroni correction for multiple comparisons when needed.P values < 0.05 were considered significant. ",
"section_name": "Statistical Analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "To evaluate the mechanistic interaction between A2AR and Notch signaling, we pre-incubated for 15 min mouse CD8+ T-cells with the selective A2AR agonist CGS-21680 prior to adding anti-CD3ε/CD28 specific antibodies.As Notch1 receptor proteolytic cleavage/activation is induced by TCR stimulation (8,10,11,30), we evaluated the levels of Notch1 receptor proteins (the transmembrane Notch1 subunit, Notch1 TM and the intracellular Notch1 domain, N1ICD) in activated CD8+ T-cells compared to unstimulated cells.Activated CD8+T-cells strongly expressed Notch1 TM and N1ICD proteins, compared to nonstimulated (NS) counterparts (Figure 1A).Notably, incubation of CD8+T cells with CGS-21680 significantly reduced the expression of both Notch1 TM and N1ICD (Figures 1B-D), suggesting that A2AR activation interferes with TCR signaling.As a control, we treated cells with the γ-secretase inhibitor (GSI) PF-3084014, which potently inhibits Notch1 cleavage (31).Incubation of cells with PF-3084014 (1 µM) prevented the generation of N1ICD following anti-CD3ε/CD28 stimulation (Figures 1B-D).Cells treated with PF-3084014 alone or together with CGS-21680 showed the highest Notch 1 down-regulation (Figures 1B-D). To further investigate the effect of the A2AR agonist on TCR-induced Notch1 signaling pathway, we determined the expression of N1ICD-target genes Hes1 (32) and cMyc (33).Hes1 and cMyc mRNA levels were reduced in CD8+T-cells treated with CGS-21680 (1 µM) and stimulated with anti-CD3ε/CD28 (Figures 1E,F, respectively).In particular, Hes1 mRNA levels upon TCR stimulation were significantly reduced 48 and 72 h after CGS-21680 treatment (Figure 1E).cMyc mRNA levels were significantly decreased at 24 and 48 h of treatment (Figure 1F).These results suggest that stimulation of A2AR decreases the expression and activation of Notch1 and N1ICD-mediated transcriptional activity in CD3ε/CD28-stimulated CD8+T-cells.The different time courses of the two transcripts may be related to different half-lives of these two transcripts or to the different mechanisms whereby N1ICD regulates the expression of Hes1 and cMyc in T-cells.Hes1 is regulated largely through a Sequence-Paired Site (SPS) closely associated with the Hes1 transcriptional start site (34), whereas cMyc is regulated primarily through a distal super-enhancer whose acetylation status is highly sensitive to depletion of N1ICD (35). To determine whether the lower levels of Notch1 protein were due to reduced mRNA synthesis, we analyzed Notch1 transcript levels in CD8+T-cells treated with CGS21680 (1 µM) and anti-CD3ε/CD28.Notch1 mRNA levels were unchanged in CD8+T-cells incubated with CGS-21680 compared to control cells (Figure 1G), indicating that A2AR stimulation decreases the levels of Notch1 protein without affecting Notch1 transcription. ",
"section_name": "A2AR Stimulation Leads to Reduced Expression and Activity of Notch1 in CD3ε/CD28-Activated CD8+T-Cells",
"section_num": null
},
{
"section_content": "To confirm that the effect of CGS-21680 on Notch1 expression was dependent on A2AR stimulation, we performed experiments in presence of the A2AR antagonist ZM-241385 (1 µM), administered in combination with CGS-21680 (1 µM).Treatment with ZM-241385 (1 µM) reversed the inhibitory effect of CGS-21680 on Notch1 expression in CD3ε/CD28stimulated CD8+T-cells (Figure 2A).To rule out off-target effects induced by the CGS-21680, we treated T cells lacking A2AR.In A2AR-/-CD8+T-cells, CGS-21680 treatment did not affect TCR-induced Notch1 levels or N1ICD levels (Figure 2B), indicating that the observed CGS-21680 effects were triggered through stimulation of A2AR.No differences in the levels of Notch2, 3, or 4 were observed in A2AR-/-CD8+T-cells (Supplemental Figure 1). ",
"section_name": "The Inhibitory Effect of CGS-21680 on TCR-Induced Notch1 Expression Depends on A2AR Stimulation",
"section_num": null
},
{
"section_content": "Stimulation of A2AR strongly inhibits the proliferation (22) and the effector functions of activated T-cells, including cytokine production (23)(24)(25)(26)(27). On the other hand, Notch1 activation promotes proliferation (8) and cytokine release (8,10,36) in CD8+T-cells.We therefore investigated the cross-talk between these signaling pathways in regulating the functional properties of CD8+T-cells, by measuring proliferation and cytokine levels in cells treated with CGS-21680 alone or in combination with GSI PF-3084014 before TCR-activation.Proliferation was assessed by flow cytometry analysis after CFSE staining.The increased CD8+ T-cell proliferation induced upon stimulation with anti-CD3ε/CD28 was slightly reduced after pre-incubation with CGS-21680 or PF-3084014 (Figure 3A).Moreover, an additive antiproliferative effect was observed after the combination of CGS-21680 plus GSI PF-3084014 (Figure 3A). Experiments were also performed in cells treated with the A2AR antagonist ZM-241385 (1 µM).Further inhibition of A2AR signaling through ZM-241385 did not affect proliferation in anti-CD3ε/CD28-activated T cells (Figure 3A). Transcript levels of granzyme B and IFN-γ were significantly reduced in cells treated with 1 µM CGS-21680 or 1 µM PF-3084014 and even more so after combination treatment with CGS-21680 + GSI before stimulation with anti-CD3ε/CD28 antibodies for 24 h (Figures 3B,D, respectively).Consistent with mRNA levels, the amounts of granzyme B and IFN-γ released from activated CD8+T-cells treated with CGS-21680 (1µM) and/or PF-3084014 (1 µM) for 72 h were significantly reduced compared to control cells (Figures 3C,E, respectively).These data indicate that A2AR stimulation and γ-secretase inhibition strongly impair the functions of activated CD8+T-cells.The effects of GCS-21680 on both granzyme B and IFN-γ were completely reversed by the A2AR antagonist ZM-241385 (1 µM) (Figures 3C,E,respectively). Treatment of A2AR-/-CD8+T-cells with CGS-21680 (1 µM) failed to reduce the release of either granzyme B (Figure 3F) or IFN-γ (Figure 3G), confirming that the suppressive effect of CGS-21680 on the release of these factors was completely dependent upon the presence of A2AR.Conversely, inhibition of γ-secretase with PF-3084014 led to a significant reduction of both granzyme B and IFN-γ release from activated CD8+T-cells from A2AR-/-mice (Figures 3F,G, respectively).These data indicate that Notch effects on granzyme B and IFN-γ release are independent of A2AR. ",
"section_name": "A2AR Stimulation and Notch1 Inhibition Strongly Reduce Proliferation and Cytokine Production in Activated CD8+ T-Cells",
"section_num": null
},
{
"section_content": "Stimulation of A2AR increases intracellular levels of cAMP in activated T-cells (22,26).Consistent with these reports, we observed that CGS-21680 treatment (1 µM) of CD3ε/CD28stimulated CD8+ T-cells increased cAMP levels within 10 min (Figure 4A).To evaluate whether increased levels of cAMP affected TCR-induced Notch1 signaling, we used the adenylate cyclase activator Forskolin (10 µM), which significantly increased cAMP levels in activated T-cells (Figure 4B).Forskolin significantly decreased the expression of Notch1 proteins induced by TCR engagement in CD8+T-cells, proliferation, production of IFN-γ and granzyme B (Figures 4C-G).When we combined Forskolin and GSI PF-3084014 (Figures 4C,D), the effect on Notch1 proteins was marked.These results suggest that cAMP-elevating agents can markedly decrease the amount of Notch1 in activated CD8+ T-cells and significantly impair Notch-dependent functions of TCR-stimulated CD8+T-cells. ",
"section_name": "Adenylate Cyclase Stimulation Mimics the Effects of CGS-21680 on Notch1 in Activated CD8+ T-Cells",
"section_num": null
},
{
"section_content": "To determine the functional role of the inhibition of Notch1 signaling in the suppressive effects induced by A2AR activation, we studied CD8+T-cells from activated-CD8+T cell conditional N1IC mice (13), which over-express Notch1IC upon activation.N1IC mice were created after crossing N1IC f/f mice with those expressing Cre Recombinase driven by Granzyme B promoter.Activated N1IC CD8+ T-cells or floxed controls (N1IC f/f ) were treated with CGS-21680 and/or GSI PF-3084014 and evaluated for proliferation or IFN-γ and granzyme B expression.Similar to the results observed in wild-type T cells, treatment of N1IC f/f cells with CGS-21680 and/or PF-03084014 slightly reduced the proliferation (Figure 5A).Conversely, activated N1IC CD8+ T-cells showed no restriction in their proliferation after treatment with CGS-21680 or PF-3084014 or a combination of both (Figure 5A), suggesting that restoring Notch1 signaling in CD8+T-cells prevents the suppressive effects induced by A2AR activation. Next, we compared the expression of IFN-γ and granzyme B in N1IC vs. N1IC f/f CD8+ T-cells treated with CGS-21680, GSI PF-3084014 or both.In agreement with the key role of Notch in the effects induced by A2AR, we found that while CGS-21680 and/or PF-3084014 reduced the frequency of IFNγ-expressing cells and the expression of granzyme B in control N1IC f/f CD8+ T-cells, they had no significant effect in N1IC CD8+ T-cells (Figures 5B,C).Overall, these results indicate that inducible expression of Notch1 intracellular domain is sufficient to overcome the suppressive effects of A2AR agonists in activated CD8+T-cells, and support the hypothesis that Notch1 is a functionally important target of A2AR-mediated suppressive effects in these cells. ",
"section_name": "Ectopic Expression of N1IC Rescues CD8+ T-Cells From the Suppressive Effects of CGS-21680",
"section_num": null
},
{
"section_content": "In CD4+ T-cells, TCR stimulation triggers Notch1 activation within 2 h with a peak at 6 h (11).This activation is mediated by a unique, Notch ligand-independent process that requires Notch endocytosis and signals from the TCR through Lck, ZAP70, phospholipase C gamma (PLCγ), a diacylglycerolactivated protein kinase C (presumably PKCθ), Ca ++ influx as well as ADAM10 or 17 and γ-secretase.PKC is activated by PDK1, in response to a PI3K co-activating signal from CD28 (37).We reasoned that if Notch1 activation in CD8+ T-cells is mediated by a similar mechanism, A2AR activation would suppress ZAP70 phosphorylation, while activation of PKC and Ca ++ influx downstream of ZAP70 would bypass A2AR-mediated Notch inhibition.Additionally, we hypothesized that A2AR agonists would be unable to block Notch1 if given after early events in TCR activation have occurred.A2AR stimulation inhibits early TCR signaling by increasing the levels of cAMP during T-cell activation.This effect is mediated by reduced ZAP70 phosphorylation (26,38).Consistent with these observations, we found that incubation of cells with CGS-21680 (1 µM) reduced Tyr319 ZAP70 phosphorylation in activated CD8+T-cells (Figure 6A).Treatment of CD8+ Tcells cells with CGS-21680 24 h after incubation with anti-CD3ε/CD28 antibodies had no effect on Notch1 protein levels (Figures 6B-D).Proliferation and release of granzyme B and IFN-γ from activated CD8+T-cells were unchanged by CGS-21680 treatment once the early TCR signal had been transduced ( Figures 6E-G, respectively). To determine whether A2AR agonists affect later stages of the TCR signaling cascade leading to Notch1 activation, we activated CD8+ T-cells with PMA and ionomycin.PMA and ionomycin induce protein kinase C (PKC) activity and Ca ++ influx (39).CD8+T-cells activated with PMA and ionomycin showed much higher levels of Notch1 TM and N1ICD compared to unstimulated cells (Figures 7A-C).In addition, treatment of these cells with CGS-21680 (1 µM) before stimulation with PMA and ionomycin had no effect on Notch1 protein levels (Figures 7A-C), suggesting that A2AR acts upstream of PKC activation during TCR-induced Notch activation.Taken together, these results indicate that the suppressive effects of CGS-21680 through A2AR on Notch1 activation rely in its capacity to block early TCR signaling events upstream of PKC activation, most likely ZAP70 phosphorylation.These effects are lost once TCR signals have been transduced and the first wave of N1ICD generation has occurred. ",
"section_name": "A2AR Stimulation Inhibits TCR-Induced Notch1 Activation/Expression by Blocking Proximal TCR Events",
"section_num": null
},
{
"section_content": "In the present study we describe a previously unknown role for the A2A adenosine receptor pathway in the control of TCRinduced Notch1 receptor activation in CD8+T-cells.Stimulation of A2AR decreases the levels of Notch1 receptor protein stabilized by TCR ligation, as well as the levels of cleaved active intracellular domain generated in CD3ε/CD28-stimulated CD8+ T-cells.This effect requires A2AR and is mediated by inhibition of TCR signaling upstream of PKC activation. Consistent with published results (22,26), activation of A2AR increases intracellular levels of cAMP.cAMP-dependent protein kinase A (PKA) phosphorylates and activates the Cterminal SRC kinase (CSK), which in turn phosphorylates and inactivates Lck, reducing tyrosine phosphorylation of the TCR/CD3ζ chain during T-cells activation (40).Lckmediated tyrosine phosphorylation of the TCR/CD3 ζ chain after T-cell activation is required for the recruitment to CD3 and activation of the zeta-chain-associated protein kinase 70 (ZAP70) and subsequent activation of other substrates that initiate downstream signals (40).In line with previous data (26,41), stimulation of A2AR with CGS-21680 reduced ZAP70 phosphorylation.Therefore, we propose a mechanism whereby stimulation of A2AR, by attenuating early TCR transduction signaling in a cAMP-dependent manner, can prevent the generation of the Notch1 intracellular domain (N1ICD) induced by TCR signaling in CD8+T-cells (Figure 8).This working model could apply not only to A2AR but also potentially to other immune suppressive molecules that signal through cAMP and PKA in T-cells, such as prostaglandin E2 (42,43). Our data indicate that A2AR signals act upstream of a diacylglycerol-dependent PKC and Ca ++ , as PMA and ionomycin bypass the effect of A2AR activation upon Notch1.Recent data show that TCR-induced, Notch ligand-independent Notch1 cleavage in T-cells requires the activation of one or more PKC isoforms, which facilitate ADAM activity (11).Previous reports indicate that the activities of ADAM10 and 17, involved in the S2 Notch1 cleavage (44) are regulated by PKCα and PKCθ (45,46).Activation of PKC is a downstream event of the TCR signaling, which depends on diacylglycerol produced by phospholipase C (PLC)-γ.PLC-γ activation in turn is triggered by the phosphorylation of ZAP70 (47).The stable activation and membrane translocation of PKCθ requires Vav and phosphatidylinositol 3-kinase (PI3K) activation, triggered by the CD28 costimulatory signal, in addition to TCR/CD3 signal (47).PKCθ, a Ca ++ -independent, phospholipid-dependent PKC isoform, is a central player in the immunological synapse, responsible for the activation of AP-1 and NF-κB downstream of the TCR (48).In this study, we observed that activation of CD8+T-cells with PMA and ionomycin, which directly and potently stimulate the activity of PKCs (47), increases the levels of Notch1 and consequently its activity.This is similar to what was described by Steinbuck and collaborators (11) The effect of CGS-21680 on Notch1 expression is A2ARdependent, since it was completely reversed by a selective A2AR antagonist and lost in CD8+T-cells from A2AR-/-mice.A2AR activation with CGS-21680 markedly inhibits the production of IFN-γ and granzyme B released from CD3ε/CD28-stimulated CD8+T-cells.These data are consistent with previous studies describing a critical role of A2AR in controlling CD8+Tcell activation in a cAMP-dependent manner, by suppressing proximal TCR signaling events (18,53).These effects as well were completely lost in activated CD8+T-cells from A2AR-/-mice, as well as in wild-type cells incubated with CGS-21680 after TCR stimulation. In CD4+ T-cells, Notch augments NF-κB activity after TCR activation (36).Moreover, Notch1 is required for CD8 effector functions, including production of IFN-γ and granzyme B (10).GSIs suppress production of IFN-γ and granzyme B (30).Consistent with published observations, GSI PF-3084014 did decrease production of IFN-γ and granzyme B in CD8+ Tcells.Our data show that simultaneous inhibition of Notch and stimulation of A2AR profoundly impair the responses of activated CD8+T-cells, including proliferation and cytokine production.This suggests that in tumor microenvironments rich in adenosine and other immune-suppressive mediators, GSIs may suppress anti-tumor CD8+ responses.GSIs have multiple targets (14), including Notch2, which is also induced by TCR stimulation in T-cells although to a lesser extent than Notch1 (54).Our data show that CD8+ T-cells ectopically expressing N1IC are remarkably resistant to the inhibitory effects of A2AR stimulation compared with controls N1IC f/f cells.These results strongly indicate that intracellular levels of Notch1 regulate the sensitivity of CD8+ T-cells to A2AR agonists, and that inhibition of Notch1 is a functionally relevant mechanism whereby A2AR agonists suppress CD8+ T-cell activation.Whether A2AR antagonists can protect Notch activity in CD8+ T-cells in tumors remains to be determined.Further investigations are also required to understand whether the A2AR signaling suppresses responses in other T-cell subsets, including CD4+T-cells and memory CD4+ or CD8+ T-cells, through inhibition of Notch. Altogether, our data suggest a new role for A2AR in blunting Notch activity, which is essential to the effector function of CD8+T-cells.We propose that the inhibitory effects of CGS-21680 on activated CD8+ T-cells are, at least partially, dependent on reduced Notch1 protein and activity after TCR stimulation.This novel cross-talk between two critical pathways offers new insights into the mechanisms whereby elevated adenosine concentrations can hamper immune cell activation in tumors and/or in inflamed injured tissues. ",
"section_name": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "We thank M. Sitkovsky and S. Hatfield for providing A2AR-/-T cells. ",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
},
{
"section_content": "This work was supported in part by NCI P01CA166009 (LM, FH, AP, BO, and LMM), U54 GM104940, \"Louisiana Clinical and Translational Science Center, \" National Institute of General Medical Sciences (LM), and P.O.R. Campania FESR 2007-2013-O.O.2.1-OCKEY (SM).CS was supported by P.O.R. Campania FESR Q72007-2013-O.O.2.1-OCKEY. ",
"section_name": "FUNDING",
"section_num": null
},
{
"section_content": "All animal studies were carried out in Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)accredited facilities in the context of protocols approved by the Institutional Animal Care and Use Committees of LSUHSC-New Orleans, the Moffitt Comprehensive Cancer Center and Northeastern University, respectively. SM and LM conceived the study, supervised experiments, and prepared the final version of the manuscript.CS, FH, AP, and RS performed experiments and interpreted their results.PR supervised experiments in Notch1IC-transgenic T-cells, which were performed by RS.SH provided A2AR-/-T cells and was responsible for maintaining adenosine receptor transgenic mouse lines.BO and LMM provided expertise on TCR signaling and PKC activity, contributed to result interpretation, reviewed and edited the final version of the manuscript. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2019.00162/full#supplementary-material 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": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "All animal studies were carried out in Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)accredited facilities in the context of protocols approved by the Institutional Animal Care and Use Committees of LSUHSC-New Orleans, the Moffitt Comprehensive Cancer Center and Northeastern University, respectively. ",
"section_name": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "SM and LM conceived the study, supervised experiments, and prepared the final version of the manuscript.CS, FH, AP, and RS performed experiments and interpreted their results.PR supervised experiments in Notch1IC-transgenic T-cells, which were performed by RS.SH provided A2AR-/-T cells and was responsible for maintaining adenosine receptor transgenic mouse lines.BO and LMM provided expertise on TCR signaling and PKC activity, contributed to result interpretation, reviewed and edited the final version of 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/fimmu.2019.00162/full#supplementary-material ",
"section_name": "SUPPLEMENTARY MATERIAL",
"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
}
] |
10.1038/s41598-020-75364-3
|
Time to first treatment and P53 dysfunction in chronic lymphocytic leukaemia: results of the O-CLL1 study in early stage patients
|
<jats:title>Abstract</jats:title><jats:p>Chronic lymphocytic leukaemia (CLL) is characterised by a heterogeneous clinical course. Such heterogeneity is associated with a number of markers, including <jats:italic>TP53</jats:italic> gene inactivation. While <jats:italic>TP53</jats:italic> gene alterations determine resistance to chemotherapy, it is not clear whether they can influence early disease progression. To clarify this issue, <jats:italic>TP53</jats:italic> mutations and deletions of the corresponding locus [del(17p)] were evaluated in 469 cases from the O-CLL1 observational study that recruited a cohort of clinically and molecularly characterised Binet stage A patients. Twenty-four cases harboured somatic <jats:italic>TP53</jats:italic> mutations [accompanied by del(17p) in 9 cases], 2 patients had del(17p) only, and 5 patients had <jats:italic>TP53</jats:italic> germ-line variants. While del(17p) with or without <jats:italic>TP53</jats:italic> mutations was capable of significantly predicting the time to first treatment, a reliable measure of disease progression, <jats:italic>TP53</jats:italic> mutations were not. This was true for cases with high or low variant allele frequency. The lack of predictive ability was independent of the functional features of the mutant P53 protein in terms of transactivation and dominant negative potential. <jats:italic>TP53</jats:italic> mutations alone were more frequent in patients with mutated IGHV genes, whereas del(17p) was associated with the presence of adverse prognostic factors, including CD38 positivity, unmutated-IGHV gene status, and NOTCH1 mutations.</jats:p>
|
[
{
"section_content": "Influence of the TP53 status on the TTFT in O-CLL1 patients.Next, the influence of the TP53 status on TTFT was evaluated in the O-CLL1 cohort.After a median follow-up of 86 months, TTFT data were available for 462/469 patients, including the 5 cases with a TP53 germline variant.The TTFT of patients with somatic TP53 mutations (Mut = Mut/noDel + Mut/Del: 24 patients) was not statistically different from that of cases with wild-type TP53 (WT/noDel: 431 patients) (HR = 1.5, 95% CI 0.8-2.7,P = 0.7).In contrast, the TTFT was significantly shorter when patients with del(17p) (Del = Mut/Del + noMut/Del: 11 patients) were compared to those without deletion (noDel: 446 patients, excluding the 5 with TP53 germline variant) (HR = 5.2, 95% CI 2.4-11.2,P < 0.0001).A multivariate analysis, in which only the presence of del(17p) and of TP53 mutations were introduced, indicated that the presence of the del(17p) at diagnosis constituted a significant risk factor for a shorter TTFT (HR 6.1, 95% CI 2.2-17.0,P = 0.001), independently of the presence of a TP53 mutation (HR 0.8, 95% CI 0.4-1.8,P = 0.6).The cohort analysed was also stratified according to the presence/absence of TP53 mutations and/or del(17p) in different combinations (see Fig. 1).The 9 Mut/Del and the 2 WT/Del patients had a significantly shorter TTFT than the 431 WT/noDel cases, whereas a somatic TP53 mutation in the absence of del(17p) (Mut/noDel: 15 patients) was not predictive of a shorter TTFT.A similar result was obtained when the five cases with germ-line allelic variants were compared with the WT TP53 cases.Subsequently we addressed the issue of whether the VAF in the Mut/noDel cases could have an influence on the prediction of the TTFT.To this end Mut/noDel cases were stratified according to high (> 10%) and low (< 10%) VAF values and the TTFT of the two groups was analysed.No significant differences were detected in the TTFT in the two groups of patients.The clinical impact of TP53 mutations with different VAF also was analysed in the group of cases with mutated IGHV genes to eliminate a possible confounding factor represented by a strong predictor of prognosis such as IGHV gene mutation status.In this group, there were 9 patients with a VAF > 10% and 4 patients with a VAF < 10%.However, no statistically significant differences in the TTFT were observed (P = 0.5) (Supplementary Fig. 1).Therefore, the size of the subclone characterised by the presence of a TP53 mutation did not appear to influence the TTFT in Binet stage A CLL.The presence of del(17p), although mostly associated with a TP53 mutation, appears to be more deleterious than the presence of the a TP53 mutation itself in determining a shorter TTFT. Functional studies on mutant P53 proteins from O-CLL1 patients.Next, the functional properties of the P53 proteins encoded by the TP53 mutations detected in our cohort were investigated.A well-established yeast-based functional assay was employed, using four reporter strains.These strains have a different P53 Response Element (RE), which is derived from the promoter of one of four human P53 effector, i.e.P21, BAX, MDM2, and PUMA, respectively.The residual transactivation ability of the mutant P53 proteins compared to that of the WT protein (set as the 100% value) was measured at 30 °C and 37 °C (Table 2).With a threshold of residual WT P53 activity of 20%, all mutant P53 proteins (excluding those encoded by TP53 germline variants) were found inactive at 37 °C, whereas some activity was observed at 30 °C (Table 2; Supplementary Fig. 2A). In fact, five mutants encoded P53 proteins with residual activity (p.Ala161Thr, p.Pro191del, p.Arg213Leu, and p.Arg282Trp within the Mut/noDel group and p.Val272Leu within the Mut/Del group).Of note, the P53 protein encoded by all TP53 germline variants (p.Asn235Ser, p.Arg283Cys, and p.Gly360Val) behaved similarly to the WT P53 and were active at both 30 °C and 37 °C (Supplementary Table 3).Mutant P53 proteins were also transiently expressed in HCT116 TP53 -/-cells and were tested for their ability to transactivate the luciferase reporter gene carrying a fragment of the P21 promoter (Supplementary Fig. 2B).The results obtained were consistent with those obtained in yeast at 30 °C, confirming that some mutants (e.g.p.AlaA161Thr, p.Pro191del and p.Arg282Trp) had a partial activity.Finally, the TP53 germ-line allelic variants identified in our cohort were found to encode proteins with a high functional efficiency of transactivation (p.Asn235Ser, p.Arg283Cys, and p.Gly360Val) in both mammalian cell-and yeast-based assays (Supplementary Fig. 2B and Supplementary Table 3). It is known that certain mutant P53 proteins have the capacity of inhibiting the activity of the WT protein when heterozygous at TP53 locus [i.e. they are Dominant Negative (DN)] 26 .Therefore, the DN potential of each mutant P53 protein of the Mut/noDel group was determined using one of the reporter strains (i.e.yLFM-P21-5′) (Table 3).None of the mutant P53 proteins with partial residual function (i.e.p.Ala161Thr, p.Pro191del, p.Arg213Leu, p.Val272Leu and p.Arg282Trp) exhibited DN potential (Table 3).Of note, all of the TP53 germline variants identified in patients without del(17p) encoded a recessive mutant P53 (Supplementary Table 4). ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Given the functional heterogeneity of P53 proteins in our cohort, we asked whether such heterogeneity could influence the TTFT.This analysis was carried out on the Mut/noDel patients' group (15 cases).Neither the transactivation ability (Fig. 2A) nor the DN potential (Fig. 2B) appeared to affect the Kaplan-Meier curves of these patients' groups, which were similar to those of the WT/noDel patients (431 patients).Although potentially interesting, these tests could not be carried out in the Mut/Del patients' group since the residual transactivation activity was < 20% in 8/9 available cases and ≥ 20% in a single case.Thus, this issue warrants re-evaluation in a larger study sample. ",
"section_name": "Influence of the residual transactivation ability and DN potential of mutant P53 proteins on TTFT in O-CLL1 patients.",
"section_num": null
},
{
"section_content": "CD38 and ZAP-70 expression, β2-microglobulin (β2M) serum levels, Rai stage, IGHV mutational status, del(11q), NOTCH1 and SF3B1 mutations, MBL classification were confirmed to have a significant prognostic power on univariate analysis 27 (Supplementary Table 5).A Cox multivariate analysis was then performed by introducing into the model markers indicative of TP53 alterations together with the variables with a significant prognostic power for TTFT determined in the univariate analysis.Both del(17p) together with TP53 mutations, and del(17p) alone, retained an independent prognostic power, whereas TP53 mutation alone did not.Other markers with an independent prognostic power were IGHV mutational status, del(11q), NOTCH1 mutation, β2M levels, and Rai stage (Fig. 3). Changes in the TP53 alterations pattern with time.TP53 mutational status was re-assessed in 171 patients after 36 months from diagnosis or at the time of therapy need.Of these, 140 re-tested at 36 months after diagnosis, did not present any new TP53 alteration.Another 23 cases, without TP53 alterations at diagnosis, were retested at the time of therapy need; again, no new TP53 alterations were recorded.Eight cases with a TP53 mutation at diagnosis were re-tested at 36 months (6 cases) or at the time of therapy need (2 cases).All of the 8 cases with a somatic TP53 mutation continued to express the same mutation seen at diagnosis, although with some changes in their VAF (Fig. 4).The 163 patients, negative for somatic TP53 mutations at diagnosis, still presented WT TP53 gene (or the same germline TP53 allelic variant) after 36 months.None of these 161 patients harboured del(17p) at diagnosis or at re-testing.The two cases re-tested at the time of therapy need presented p.Arg175His or p.Val157Asp, respectively.Of these, one [with p.Arg175His, and a del(17p) both at diagnosis and at re-testing] exhibited an over fourfold VAF increase, while the other [with p.Val157Asp mutation and no del(17p) also at re-testing] did not manifest any change in VAF. ",
"section_name": "Multivariate analysis.",
"section_num": null
},
{
"section_content": "The present study investigated the influence on disease progression of TP53 alterations in Binet A CLL patients, enrolled in the multicentre prospective observational O-CLL1 trial with a long follow-up (median ~ 7 years).This influence was measured by determining the TTFT, which represents a rather accurate approach to assess disease progression.TP53 mutations and del(17p) were determined in 98.7% of patients (469/475).There are differences in the thresholds distinguishing positive from negative cases for the FISH and NGS analysis (i.e.measurement of del(17p) and TP53 mutations, respectively) due to the different sensitivity of each methodology.Since the sensitivity is somewhat imbalanced in favour of NGS, it cannot be excluded that certain patients harbour a del(17p), in addition to the TP53 mutation, but that the former lesion goes undetected by FISH being present at sub-threshold level in a small sub-clonal component.Therefore, all the categories of TP53 alterations dealt within the present paper refer to those detected within these technical limits.This admittedly may represent a limitation, especially in cases with VAF < 10%. The overall incidence of somatic TP53 mutations [5% (24/469 patients)] and del(17p) [2.3% (11/469 patients)] in the O-CLL1 cohort and their relative distribution are similar to that of previous reports 4,16,17,21 .The majority of TP53 mutations were point mutations, causing a single amino-acid substitution in the P53 protein DNA binding domain.This was true for both the Mut/noDel and the Mut/Del patient groups.Although over 2,000 amino acid substitutions caused by a point mutation in the TP53 gene have been identified, 3 amino acid substitutions (p.Arg175His, p.Pro278Arg, and p.Ile195Thr) were shared by the Mut/noDel and Mut/Del patients' groups. The biological features of Mut/Del and Mut/noDel CLL patients were significantly different, since adverse prognostic factors, such as CD38 positivity (P = 0.006), UM-IGHV gene status (P = 0.003), and NOTCH1 Table 2. Evaluation of the transactivation ability of somatic TP53 mutations using a yeast-based assay.Results are shown as residual activity of the mutant P53 protein with respect to wild-type (WT) P53 protein set as 100%.Each single mutant P53 was expressed in four different reporter yeast strains identified by the P53 Response Element (RE) from the promoter of the P21, PUMA, MDM2 or BAX effector genes.Transactivation ability was determined by growing yeast at 30 °C and 37 °C. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "mutations (P = 0.0415), were less frequently observed in the Mut/noDel patients' group.We also observed that the TTFT of patients with mutated or WT TP53 gene was similar (P = 0.7), a finding which is in line with that of Brieghel et al. 28 , who demonstrated that TP53 mutations, determined by NGS, did not influence the clinical course of patients, in the absence of del(17p); this was true for cases with high (VAF ≥ 10%) and low (VAF < 10%) TP53 mutation burden 28 .We also did not find differences in the TTFT of Mut/noDel patients when they were stratified into two groups based upon high or low VAF, and this was also true for the patients with TP53 mutations from the mutated IGHV group stratified according to VAF values.Therefore, the size of the cell sub-clone bearing the TP53 mutations does not appear to influence disease progression for Binet stage A CLL patients.In this study, the TTFT of the patients with del(17p) was significantly shorter than that of patients without deletion (P < 0.0001), while del(17p) concomitant with a TP53 mutation represented, an additional and independent prognostic factor associated with shorter TTFT in multivariate analyses.However, in interpreting these data, it should be noted that most patients with del(17p) also had a TP53 mutation, and that only two cases had del(17p) without a TP53 mutation.Therefore, it is difficult to precisely evaluate the contribution of del (17p) alone to disease progression with this low number of cases.However, it is of note that Yu et al. 29 reported that Mut/Del CLL patients had a TTFT shorter than that of the Mut/noDel patients.Moreover, Hoechstetter et al. 30 showed that del(17p) is the highest weighted factor of the six considered in a multivariate analysis to predict TTFT and OS in Binet stage A CLL, although it requires the cooperation of additional factors to determine progression.Unlike that reported in this and in the other studies quoted above, Dicker et al. 18 and Rossi et al. 5 reported that TP53 mutations alone were capable of predicting a shorter TTFT.These discrepancies are difficult to explain also given the differences in methodologies used, clinical study design, and cohorts investigated.However, it is worth underlining that a predominance of Binet B and C stage patients was included in those studies, whereas we focused on Binet A cases only. The mutant P53 proteins encoded by TP53 mutations detected in this study were investigated further for their transactivation ability and DN potential.Although mutant P53 proteins appeared to be functionally heterogeneous, such heterogeneity was not associated with differences in TTFT within the Mut/noDel patients' group.However, the interpretation of these data requires caution because of the relatively low number of Mut/noDel cases and further analyses in larger cohorts seem to be needed.Furthermore, it is worth recalling that mutant P53 proteins can acquire new functions favouring tumour cell expansion.These properties, indicated collectively as gain of function 24 , may in principle be present in CLL patients and perhaps have a higher incidence in more advanced cases.These too deserve analyses in large cohorts of patients. IGHV mutational status represents a valuable prognostic marker for risk progression and outcome 11 .In the O-CLL1 cohort, the majority of cases with del(17p) had unmutated IGHV genes, whereas the majority of Mut/ noDel cases had mutated IGHV genes.This finding may concur to explain why Mut/noDel cases progressed more slowly to the more advanced stages requiring therapy (Table 1).Furthermore, Mut/noDel cases were mostly negative for other unfavourable prognostic markers, whereas this was not the case for the group of patients with del(17p) (Mut/Del and WT/Del) (Table 1).These considerations may also help explain the discrepancies in the prognostic role of TP53 mutations between this study and those including Binet B and C patients.Since unmutated-IGHV cases are likely to progress more rapidly towards advanced stages 31,32 , it is possible that cases with mutated IGHV genes and TP53 mutations, that may not progress, are less frequently found in cohorts comprising numerous advanced cases. Re-evaluation of 171 patients from this study following a 36 months interval or at the time of therapy need did not reveal any changes in TP53 alterations status.Specifically, there was neither an acquisition/loss of a TP53 mutation nor of del(17p).Only in one patient an expansion of a sub-clone carrying a non-functional TP53 mutation was observed at progression. In conclusion, the present study based on a clinically and molecularly well-characterised Binet stage A CLL cohort demonstrates that the occurrence of del(17p) significantly predicted TTFT, while that of a TP53 mutation alone, was unable of such prediction. ",
"section_name": "ID",
"section_num": null
},
{
"section_content": "All methods were carried out in accordance with relevant guidelines and regulations. ",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "therapy according to NCI guidelines were prospectively enrolled within 12 months of diagnosis (O-CLL1 protocol, clinicaltrial.govidentifier NCT00917540).This protocol was presented by the Gruppo Italiano Studio dei Linfomi (GISL) on behalf of several Italian participating institutions and approved by the Ethics Review Committee (Comitato Etico Provinciale, Modena, Italy).Written informed consent was obtained from all patients in accordance with the declaration of Helsinki.The ethics committees from each participating centre (listed in the acknowledgements) approved this study.The median time between diagnosis and patient enrolment in the study was 2.3 months.A total of 420 (89.6%) and 386 (82.3%) patients had follow-up data at 1-and 2-year, respectively.Recruitment began in January 2007 and the criteria for CLL diagnosis employed followed the 1996 NCI/WG guidelines requiring > 5000 lymphocytes/µL in the peripheral blood.One hundred thirty-six cases (26%) fulfilled the definition of MBL (i.e.< 5.0 × 10 9 B lymphocytes/L in the peripheral blood and no apparent lymph node, spleen, or liver enlargement) according to the more recent NCI/IWCLL classification 33 .Treatment was decided uniformly for all participating centres based on documented progressive and symptomatic disease according to National Cancer Institute-sponsored working guidelines 33 .CLL cell phenotypes, CD38, and ZAP-70 expression, and IGHV mutational status assessment was centralised in the laboratory in Genoa, while all FISH assays were performed in Milan. The median age of the entire cohort was 61.2 years, 214 cases (41%) were female.Finally, at the time of the present analysis, 179 cases (35.2%) progressed and were treated. Ethical parameters.Ethical parameters, included in the synopsis of O-CLL1 protocol, clinicaltrial.govidentifier NCT00917540 were the following: It is responsibility of the investigator(s) to submit a copy of the protocol and detailed patient information sheet-consent form to an Independent Ethics Committee or Institutional Review Board in order to obtain independent approval to conduct the study.It is responsibility of the investigator(s) to ensure that the study is conducted in full conformance with the principles of the current version of the Declaration of Helsinki and to ensure that the study is performed in accordance with the international Good Clinical Practice (GCP) standards and according to all local laws and regulations concerning clinical studies). ",
"section_name": "Patients and CLL cells preparation. Only previously untreated Binet stage A CLL patients not requiring",
"section_num": null
},
{
"section_content": "Peripheral blood mononuclear cells (PBMCs) from patients with CLL were isolated by Ficoll-Hypaque (Seromed, Biochrom) density gradient centrifugation.CD19-positive CLL cells were enriched by negative selection with the EasySep-Human B-cell Enrichment Kit without CD43 depletion (STEM-CELL Technologies, Voden Medical Instruments S.p.A.), using the fully automated protocol of immunomagnetic cell separation with RoboSep (Stem Cell Technologies).The percentage of purified B Cells (CD19+) exceeded 95%, as detected by flow cytometry 34 . CD38 and ZAP-70 determination, FISH analyses, NOTCH1 and SF3B1 mutations, and IGHV gene analysis.Heparinized blood samples were obtained and immediately shipped to the Genoa laboratory at room temperature, to arrive on the same or the following day; the cells were immediately processed.In these conditions, repeated quality control tests indicated minimal cell apoptosis or necrosis as measured by flow cytometric analysis using annexin V or propidium iodide (PI) staining.CD38 positive leukemic cells were measured by triple staining with CD19 fluorescein isothicyanate (FITC), CD38 phycoerythrin (PE), and CD5 Cy-Chrome (Becton Dickinson & Co., Sunnyvale, CA, USA).The cells were analysed using a FACS Calibur flow cytometer (Becton Dickinson & Co.) as previously described 35 .ZAP-70 was determined by flow-cytometry.CLL cells were first incubated with CD3 PE-CY7, CD19 PE and CD5 allophycocyanin (APC) monoclonal antibodies (mAbs) (Becton Dickinson & Co.), fixed, permeabilised with Fix and Perm reagents (Caltag Laboratories) and exposed to a ZAP-70 FITC (Upstate, Lake Placid, NY, USA) or an isotype control mAb (mouse IgG2a FITC; Becton Dickinson).Cytogenetic abnormalities involving deletions at chromosomal loci 11q22.3,13q14.3,17p13.1, and trisomy 12 were evaluated by FISH in a purified CD19+ population, as previously described 36 .The FISH study was performed using the protocol provided by the manufacturer of the multicolour probes LSI D13S25/LSI 13q34, LSIp53/CEP17, LSI ATM/CEP11, and CEP12 (Abbot Park, IL, USA).A total of 200 interphase nuclei were analysed for each probe set.The cut-off points (mean + 3 standard deviations) for positive values assessed on peripheral mononuclear cells from ten control subjects were 3.4%, 1.7%, 3.8% and 3.4% for + 12, del(11q), del(13q), and del(17p), respectively 36 . IGHV mutational status was assessed using cDNA, as previously described 37 .Sequences were aligned to the IMGT directory and analysed using IMGT/VQUEST software.SF3B1 (exons 14, 15, and 16, including splice sites; RefSeq NM_012433.2) genes were analysed by PCR amplification and Sanger sequencing of high molecular weight genomic DNA extracted from CD19+ purified B-cells (> 95%) as previously described 38 .The NOTCH1 c.7541_7542delCT mutation was tested by NGS using Roche 454 technology and subsequently validated by Amplification Refractory Mutation System (ARMS)-PCR as previously described 39 . Evaluation of TP53 mutations.The TP53 mutational status of all cases (n = 475) was analysed by NGS using Roche Junior (Roche-454 Life Sciences, Penzberg, Germany) in 250 cases as previously described 40 and by the Ion Torrent platforms (Thermo Fisher Scientific Carlsbad, CA) in 241 cases (see https ://tools .thermofish er.com/conte nt/sfs/manua ls/MAN00 13432 _Ion_Ampli Seq_Libra ry_Prep_on_Ion_Chef_UG.pdf for further details).Sixteen cases were tested using both platforms with 100% concordance.Briefly, TP53 libraries were prepared using genomic DNA extracted from CD19+ purified B cells (QIAamp DNA Blood Mini Kit, Qiagen Hilden, Germany) according to the respective protocols (see above) and then sequenced with both NGS platforms taking into account the achievement of a threshold of 500 reads for each TP53 amplicon.Cases with VAF ≥ 2% were considered positive for TP53 mutation; in addition, samples with TP53 variant < 10% VAF were re-evaluated by a second NGS run.The panel utilized for the Ion torrent NGS study was the Ion AmpliSeq™ TP53 Panel comprising 24 primer pairs across 2 pools that provides 100% of coverage of exons 2-11 and exon-intron boundaries (± 30 bp padding).The TP53 primer panel applied to Roche NGS technology spanned exons 4 to 9 40 .Of the 171 cases sequenced (131 with Roche J, 51 with Ion Torrent, 11 were subsequently analysed using both sequencing approaches with concordant results), TP53 status was determined not only at the time of diagnosis, but also after 36 months or at the time of therapy need.Any TP53 variant identified [exonic, intronic and frequent SNPs (e.g.Pro72Arg)] identified by NGS, was recorded for each sample.For all samples in which the variant was verified, and the VAF was approximately 50%, suggesting a germline origin, the patient-matched normal tissue DNA was subsequently analysed by NGS.For patients CG0015 and RA0023, DNA was obtained from buccal swabs (3 separate swabs per patient).For GS0473, RC0479, and NF0056 non-tumour DNA was obtained from CD3+ cells obtained by triple staining with CD19, CD5, and CD3 mAbs (Becton Dickinson) followed by cell sorting (FACS ARIA II, Becton Dickinson) of CD3+ CD5+ cells to avoid any contamination of CD19+ CD5+ neoplastic cells 10 .No mutations in exons 2, 3, or 11 were identified by Ion Torrent NGS among the 241 CLL cases analysed using this platform, while in only one case (1/241, 0.4%) a germline variant, was discovered in exon 10.This suggests that there was a number of mutated samples, possibly not being detected by sequencing of exons 4-9 with the Roche platform, that was negligible or non-existing, and that the results obtained with the two methods were comparable.Overall, sequencing of the entire coding portion of the gene using the Ion AmpliSeq™ TP53 Panel indicated that exons 4-8 were the most recurrently mutated.The functional analysis, performed using a yeast-based assay, was applied, to those TP53 variants causing either amino-acid substitutions or coding for a truncated P53 protein. ",
"section_name": "CLL cells preparations.",
"section_num": null
},
{
"section_content": "Yeast strains and media.The yLFM-P21-5′, yLFM-PUMA, yLFM-MDM2P2C, and yLFM-BAX A + B yeast strains were used to assess the functionality of TP53 variants: all strains were isogenic except for the different P53 response element (RE) located upstream of the luciferase reporter gene (LUC1).Cells were grown in YPDA medium (1% yeast extract, 2% peptone, 2% dextrose, 200 mg/L adenine) or in selective medium (with or without 2% agar) containing dextrose or raffinose as a carbon source plus adenine (200 mg/L), but in the absence of tryptophan and/or leucine (Sigma-Aldrich, Saint Louis, Missouri, USA; Biokar Diagnostics, Allonne, France).Galactose (Sigma-Aldrich, Saint Louis, Missouri, USA) was added to the medium to modulate P53 expression under the inducible GAL1,10 promoter. Yeast vectors.For the transactivation assay, human WT and mutant P53 proteins were expressed using a pTSGbased vector (TRP1).The P53 mutants were constructed in the pTSG-based vector (through SgraI/StuI digestion and subsequent ligation) (New England Biolabs) from available pLS-based vector 26,41 .When cloned mutants were not available, P53 mutants were firstly constructed in a pLS-or pTS-based vector exploiting in vivo yeast homologous recombination, as previously described 42 and then cloned (through SgraI/StuI or XhoI/NotI digestion and subsequent ligation) (New England Biolabs) in the pTSG-based vector. For the dominance assay, P53 mutant protein was expressed using a pLS-or pTS-based vector; the pLS89 (TRP1) or pLLS89 (LEU2) expressing WT P53 protein under the inducible GAL1,10 promoter was co-transformed in yeast, based on the previously used selection marker.Plasmid pRS314 (TRP1) and pRS315 (LEU2) were used as empty vectors.The list of primers used to construct pLS-or pTS-based P53 mutant vectors is available upon request. Yeast functional assay.Quantitative functional assays (evaluation of transactivation ability and DN potential) were performed according to the miniaturized protocol we developed 43 .The transactivation activity of a mutant P53 was measured by calculating the percentage with respect to WT P53 (set as 100%); the DN potential was calculated by comparing the net activity of WT and mutant P53 co-expression with respect to the expression of WT P53 alone (set as 100%).A mutant P53 was defined as recessive or dominant when the net activity was above or below 100%, respectively. Evaluation of P53 function in a mammalian reporter-assay.Cell line and media.HCT116 TP53 -/- cells (human colon carcinoma) were obtained from Dr. B. Vogelstein (The Johns Hopkins Kimmel Cancer Center, Baltimore, MD).Cells were grown in RPMI containing 10% foetal bovine serum, L-glutamine, and a penicillin-streptomycin antibiotic mixture (Euroclone, Milano, Italy), and maintained at 37 °C in 5% CO2 at 100% humidity. ",
"section_name": "Evaluation of P53 function in a yeast-reporter assay.",
"section_num": null
},
{
"section_content": "A pCIneo-based (Promega) vector was used to express WT and mutant P53 in mammalian cells.Plasmids expressing mutant P53 were constructed as previously described 42 .The pGL3-1138 and the pRL-SV40 plasmids were used as reporter (P21 promoter) and normalization vectors, respectively 44 . Mammalian functional assay.HCT116 TP53 -/-cells transfected with pCI-neo-based P53 expression vectors, the reporter, and normalization plasmids, were collected and washed with cold PBS.Lysis was performed in 1X PLB buffer (Passive Lysis Buffer, Promega).Luciferase assays were conducted as previously described 45 . Statistical analysis.Statistical analyses were performed as previously described 25,27 .Briefly, SPSS for Windows, v13.0, 2004 software (SPSS, UK) was used for all the analyses.We performed, statistical comparisons, for categorical variables, using two-way tables for the Fisher's exact test, while multiway tables were used for the Pearson's Chi-square test.TTFT analyses were performed using the Kaplan-Meier method in patients with a minimum follow-up.Using the log-rank test, we calculated the statistical significance of associations between individual variables and survival.We investigated the prognostic impact for the outcome variable by univariate and multiple Cox regression analysis.Data were expressed as hazard ratio (HR) and 95% confidence interval (CI).A value of P < 0.05 was considered statistically significant. ",
"section_name": "Mammalian expression and reporter vectors.",
"section_num": null
}
] |
[
{
"section_content": "In addition to the listed Authors, the following Investigators participated in this study as part of the Gruppo Italiano Studio Linfomi (GISL): Gianni Quintana, Divisione di Ematologia, Presidio Ospedaliero \"A.Perrino\", Brindisi; Giovanni Bertoldero, Dipartimento di Oncologia, Ospedale Civile, Noale, Venezia; Paolo Di Tonno, Dipartimento di Ematologia, Ospedale di Venere, Bari; Robin Foà and Francesca R Mauro, Divisione di Ematologia, Università La Sapienza, Roma; Nicola Di Renzo, Unità di Ematologia, Ospedale Vito Fazzi, Lecce; Maria Cristina Cox, Ematologia, A.O. Sant' Andrea, Università La Sapienza, Roma; Stefano Molica, Dipartimento di Oncologia ed Ematologia, Pugliese-Ciaccio Hospital, Catanzaro; Attilio Guarini, Unità di Ematologia e Trapianto di Cellule Staminali, Istituto di Oncologia \"Giovanni Paolo II\", Bari; Antonio Abbadessa, U.O.C. di Oncoematologia Ospedale \"S.Anna e S. Sebastiano\", Caserta; Francesco Iuliano, U.O.C. di Oncologia, Ospedale Giannettasio, Rossano Calabro, Cosenza; Omar Racchi, Ospedale Villa Scassi Sampierdarena, Genova; Mauro Spriano, Ematologia, A.O. San Martino, Genova; Felicetto Ferrara, Divisione di Ematologia, Ospedale Cardarelli, Napoli; Monica Crugnola, Ematologia, CTMO, Azienda Ospedaliera Universitaria di Parma; Alessandro Andriani, Dipartimento di Ematologia, Ospedale Nuovo Regina Margherita, Roma; Nicola Cascavilla, Unità di Ematologia e Trapianto di Cellule Staminali, IRCCS Ospedale Casa Sollievo della Sofferenza, San Giovanni Rotondo; Lucia Ciuffreda, Unità di Ematologia, Ospedale San Nicola Pellegrino, Trani; Graziella Pinotti, U.O. Oncologia Medica, Ospedale di Circolo Fondazione Macchi, Varese; Anna Pascarella, Unità Operativa di Ematologia, Ospedale dell' Angelo, Venezia-Mestre; Maria Grazia Lipari, Divisione di Ematologia, Ospedale Policlinico, Palermo, Francesco Merli, Unità Operativa di Ematologia, A.O.S. Maria Nuova, Reggio Emilia; Luca Baldini Istituto di Ricovero e Cura ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This work was supported by: Associazione Italiana Ricerca sul Cancro (AIRC) Grant 5 × mille n.9980, (to M.F., F.M., and A.N.); AIRC I.G. n.14326 (to M.F.), n.15426 (to F.F.), and n. 5506 (to G.F.); AIRC and Fondazione CaRiCal co-financed Multi-Unit Regional Grant 2014 n.16695 (to F.M.); Italian Ministry of Health 5 × 1000 funds 2013 (to G.F.) 2014 (to G.C, S.Z.and.A.I.), 2015 (to F.F. S.Z., and G.F.) and 2016 (to F.F., G.C. and G.F.); ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "M.L., G.DeL., S.M.M.C.S.F., M.B., S. Z., A.G.R., and M.D. performed biological characterization (including NGS sequencing and cytogenetic analysis) of CLL samples.PMo PMe, and AS were involved in the functional characterisation of mutant P53; M.G., A.I., A.N. M.F., and F.M. were involved at the clinical level; M.F. e F.M. were P.I. of the OCLL1 clinical Trial; FM performed statistical analysis; M.F., F.F., G.F., G.C. and F.M. conceived the study and discussed the results.G.F. and F.M. wrote the paper.All authors critically read the manuscript, suggested changes and approved the final version of this manuscript. The authors declare no competing interests. ",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "M.L., G.DeL., S.M.M.C.S.F., M.B., S. Z., A.G.R., and M.D. performed biological characterization (including NGS sequencing and cytogenetic analysis) of CLL samples.PMo PMe, and AS were involved in the functional characterisation of mutant P53; M.G., A.I., A.N. M.F., and F.M. were involved at the clinical level; M.F. e F.M. were P.I. of the OCLL1 clinical Trial; FM performed statistical analysis; M.F., F.F., G.F., G.C. and F.M. conceived the study and discussed the results.G.F. and F.M. wrote the paper.All authors critically read the manuscript, suggested changes and approved the final version of this manuscript. ",
"section_name": "Author contributions",
"section_num": null
},
{
"section_content": "The authors declare no competing interests. ",
"section_name": "Competing interests",
"section_num": null
}
] |
10.31557/apjcp.2020.21.9.2783
|
LTA, LEP, and TNF-a Gene Polymorphisms are Associated with Susceptibility and Overall Survival of Diffuse Large B-Cell lymphoma in an Arab Population: A Case-Control Study
|
Objective: In this study, we aimed to explore the relationship between five selected proinflammatory and immune-mediated genes (TNF rs1800629G>A, rs361525G>A, rs1799964T>C, LTA rs1800683G>A, rs909253A>G, TNFAIP8 rs1042541C>T, LEPR rs1327118G>C, and LEP rs2167270G>A) and the risk and overall survival of DLBCL patients within the Jordanian Arab population. Methods: One hundred twenty-five patients (125) diagnosed with DLBCL at the King Abdullah University Hospital (KAUH) between 2013 and 2018 and 238 healthy cancer-free control subjects with similar geographic and ethnic backgrounds to the patients were included in the study. Genomic DNA was extracted from the formalin-fixed paraffin-embedded tissues of the subjects and from peripheral blood samples of the controls. The Sequenom MassARRAY® sequencer system (iPLEX GOLD) was used. The analyses included assessments of population variability and survival. Results: Our study showed significant differences in the distribution of the studied polymorphisms of DLBCL between the patients and controls for TNF rs1800629G>A, LTA rs909253 G>A and LEP rs2167270 G>A. TNF rs1800629G>A (p = 0.01), in which the G allele harbors a higher risk of DLBCL (GG and GA genotypes when compared with AA genotype) (p = 0.044). The LTA rs909253 A>G polymorphism is associated with a higher risk of DLBCL in the allelic model (p = .004). LEP rs2167270 G>A polymorphism is associated with a decreased risk of DLBCL in the recessive mode models (p = .03). Subjects with the dominant model for TNF-a rs1799964 (TT genotype in comparison with the combined TT/TC genotype) and patients with the homozygous genotype (GG) of rs361525 have better overall survival rates. Conclusion: Our results confirmed the diversity and the heterogeneity of the disease. Although the study has a limitation because of its relatively small size, it clearly emphasizes the significance of ancestry and genetic composition as the determinants of DLBCL risk and behavior.
|
[
{
"section_content": "The World Health Organization's (WHO) classification of hematolymphoid malignancies depends on a combination of morphologic, immunophenotypic, cytogenetic, and molecular findings.(Swerdlow et al., 2016) Accordingly, mature B-cell lymphomas are divided into Hodgkin and non-Hodgkin lymphoma types (HL and NHL).NHL is one of the most common hematologic malignancies with over 60 subtypes sharing the cell of origin but ranging in behavior from indolent to aggressive and very aggressive types.NHL is considered the sixth most common type of RESEARCH ARTICLE LTA, LEP, and TNF-a Gene Polymorphisms are Associated with Susceptibility and Overall Survival of Diffuse Large B-Cell lymphoma in an Arab Population: A Case-Control Study cancer and the ninth leading cause of cancer deaths among both males and females worldwide (Siegel et al., 2016;Teras et al., 2016). The incidence of non-Hodgkin lymphoma (NHL) is increasing, and in the United States alone, approximately 81,080 new cases have been diagnosed in 2016.That comprises 89% of all new lymphoma cases.However, the trends of NHL incidence are variable and show significant age, gender, racial, and geographic differences (Kamangar et al., 2006;Horesh and Horowitz, 2014;Ferlay et al., 2015;Perry et al., 2016;Batista et al., 2017).Generally, the overall incidence of NHL is higher in males, but females show greater extra-nodal involvement (Castillo et al., 2014). Regarding geographic variation, developing countries show lower incidence but higher grade of B-cell lymphomas in comparison with developed countries (Perryet al., 2016).The surrounding environment, infectious microorganisms, and lifestyle all play an important role in NHL pathogenesis (Morton et al., 2008;Crump et al., 2014;Ollberding et al., 2014;Slager et al., 2014). Diffuse large B-cell lymphoma is the most common NHL (Harris et al., 2000).DLBCL is a diverse disease and can be classified into two genetically distinct types: the germinal center B-like type, characterized by the expression of germinal center B-cell genes, and the activated B-like type, characterized by the gene expression of activated peripheral blood B-cells.Clinically, the germinal center B-like type shows better overall survival (Alizadeh et al., 2000).The standard chemotherapy regimen for patients with DLBCL is the CHOP regimen (cyclophosphamide, doxorubicin, vincristine, and prednisone).Adding Rituximab (an anti-CD20 monoclonal antibody) increases the rate of complete remission and overall survival in comparison with the CHOP regimen alone (Coiffier et al., 2002). Several studies reveal that different genetic loci are linked with risk and/or outcome in DLBCL.Among them, the most notable genes are the IL6, IL10, LEPR, CTLA-4, IL4RA, TNF-α, and LT-a immunity genes (Berglund et al., 2005;Wang et al., 2006;Zhang et al., 2012).There is strong proof that changes in immunological functions pose an increased risk of lymphoma.Inflammatory and immune-response genes are the inventive messengers of adaptive immunity; they regulate the function of the immune system and growth of the lymphoid tissues. In this study, we aimed to investigate the relationship between eight single nucleotide polymorphisms (SNPs) in five selected proinflammatory and immune-mediated genes (TNF rs1800629G>A, rs361525G>A, rs1799964T>C, LTA rs1800683G>A, rs909253A>G, TNFAIP8 rs1042541C>T, LEPR rs1327118G>C, and LEP rs2167270G>A) and the risk and overall survival of DLBCL patients in the Jordanian Arab population. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "One hundred twenty-five (125) patients and two hundred and thirty-eight (238) healthy cancer-free control subjects with similar environmental and racial backgrounds to the patients were included in the study.All cases of DLBCL diagnosed from the beginning of 2013 to the end of 2018 were collected from the archives of pathology of King Abdullah University Hospital.All cases were reviewed by (SK) and one representative section was chosen from each case.All the procedures in the study was approved by the Scientific Research Committee and Institutional Review Board (IRB) at the Jordan University of Science and Technology [ IRB code number 5/106/2017, dated 8/06/2017] in accordance with the 1964 Declaration of Helsinki and its later amendments. For the patients; the need for formal written informed consent was waived by the IRB.All control subjects were voluntarily involved and signed written informed consent.Cases' and controls' names were coded and blinded and treated confidentially. ",
"section_name": "Patients and data collection",
"section_num": null
},
{
"section_content": "Formalin-fixed paraffin-embedded tissues of the DLBCL patients were used to extract the genomic DNA using the commercially available DNeasy Blood and Tissue Kit (Qiagen Ltd., West Sussex, UK), according to the manufacturer's protocols.Peripheral blood samples were used to extract the genomic DNA of control healthy subjects using the QIAamp® or Promega DNA Mini Kit, in accordance with the manufacturer's instructions.The quality of the extracted DNA was examined using agarose gel electrophoresis and ethidium bromide staining.The concentration and purity of the extracted DNA were assessed using a NanoDrop 1000 ® spectrophotometer.The pure DNA samples and their concentrations were sent to the Australian Genome Research Facility (AGRF, Melbourne Node, Melbourne, Australia) for the genotyping of TNF (rs1800629G>A, rs361525G>A, and rs1799964T>C), LTA (rs1800683G>A and rs909253A>G), TNFAIP8 (rs1042541C>T), LEPR (rs1327118G>C), and LEP (rs2167270G>A).Genotyping with the Sequenom MassARRAY® system (iPLEX GOLD) (Sequenom, San Diego, CA, USA) was performed at the AGRF, as per the manufacturer's recommendations (Sequenom, San Diego, CA, USA).The SNPs, SNP positions, and primer sequences for the TNF-a, LTA, TNFAIP8, LEPR, and LEP genes are shown in Table 1. ",
"section_name": "DNA analysis",
"section_num": null
},
{
"section_content": "The follow-up period was calculated by subtracting the date of diagnosis from the date of death for the dead cases and by subtracting the date of diagnosis from the last updated date for the vital status of those patients who were alive until the last date of check-up.The actuarial life table survival analysis was used to obtain the overall survival probabilities.Cox's proportional hazard regression was used to identify the independent factors related to survival.The survival analyses (genotypic, allelic, and clinical data association) were performed using the Statistical Package for the Social Sciences (SPSS), version 25.0 (SPSS, Inc., Chicago, IL).The survival curves were displayed using the GraphPad Prism 6 software.The SNPStats Web Tool was used to analyze the distribution of genotypes in the patients and the associations between polymorphisms and clinical variables (independent variables), including gender, age, and response to treatment.The patients' clinical characteristics and response rates were compared using Chi square tests.The Hardy-Weinberg equilibrium was estimated through a goodness-of-fit χ2 test.All the results were considered significant (P < 0.05). ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Table 2 summarizes patients' demographic and clinical data.One hundred and twenty-five DLBCL patients shows the distribution of both the genotypic and allelic frequencies of the eight SNPs.The allelic distribution did not reveal any significant differences in the distributions of the studied DLBCL polymorphisms between patients and controls except for TNF rs1800629 (p = o.o16) and LTA rs909253 (p = o.oo4).In our population, the patients with the G allele of TNF rs1800629 and A allele of LTA rs909253 had a higher risk of developing DLBCL in comparison with those having the A allele for TNF rs1800629 and the G allele for LTA rs909253. Additional analyses based on four genetic models (codominant, dominant, recessive, and overdominant) shows that LEP rs2167270 G>A polymorphism is associated with a decreased risk of DLBCL in the recessive mode models (p = 0.03).The results are shown in Table 4. ",
"section_name": "Demographic and Clinical Data",
"section_num": null
},
{
"section_content": "All the 125 DLBCL patients were included in the survival analysis.Survival analyses were performed using the Kaplan-Meier curve and log-rank test.The only SNPs to show significant survival results were TNF-a rs1799964 and TNF rs361525.In our study, subjects with the dominant model for TNF-a rs1799964 (TT genotype and 238 ethnically and geographically matched healthy controls were enrolled in this study.Of the patients, 66 were male and 59 were female (52.8%/47.2%)with a mean age of 53.7 years.The mean age for the controls was 43.2 years (6-89) and 38.7% consisted of male subjects.Most of the patients (74%/59.2%) at the time of diagnosis were at the Ann Arbor stage IV of lymphoma.The mean levels for LDH, total protein, and serum albumin at the first encounter were 635 U/L, 58.6 g/dl, and 35.4 g/dl, respectively.The mean percentage of monocytes in the peripheral blood was 6.9%. ",
"section_name": "Association between TNF, LTA, TNFAIP8, LEPR and LEP genes polymorphisms and the survival rate of DLBCL",
"section_num": null
},
{
"section_content": "Eight single nucleotide polymorphisms (SNPs) in four inflammation and immune-related genes (TNF rs1800629G>A, rs361525G>A, rs1799964T>C, LTA rs1800683G>A, rs909253C>T, TNFAIP8 rs1042541C>T, LEPR rs1327118C>G, and LEP rs2167270G>C) were genotyped in all the subjects (patients and controls). The genotype distributions of the patients and controls were compared.Unconditional logistic regression analysis was used to estimate the association between the genotype frequency and the risk of developing DLBCL. in comparison with the combined TT/TC genotype) and patients with the homozygous genotype (GG) of rs361525 have better overall survival-rates.Figure 1 and Figure 2. ",
"section_name": "Association between TNF, LTA, TNFAIP8, LEPR and LEP genes polymorphisms and the risk of DLBCL",
"section_num": null
},
{
"section_content": "Lymphomagenesis is a complex process involving an interaction between the tumor cells and the surrounding stromal cells or extracellular matrix (Balkwill and Mantovani, 2001).The tumor-microenvironment interaction is influenced by proinflammatory and immunerelated factors that affect the proliferation and maturation of the interacting cells (Kurzrock, 1997).There is increasing evidence that the genetic composition of the host (gene polymorphism) influences incidence variation and the outcomes of patients with a similar tumor stage and histological grade (Bagg, 2004;Wang et al., 2009;Dunleavy et al., 2014;Glass et al., 2016). Based on their ability to kill mouse fibrosarcoma L-929 cells, two cytotoxic factors were subsequently reported: the first was the lymphotoxin (LT) factor, which is produced by lymphocytes and was recognized by Gale A Garner in 1968 (Kolb and Granger, 1968).And the second was the tumor necrosis factor (TNF), discovered in 1975 by LIoyd J. Old (Nedwin et al., 1985).The last exon is similar in both LT and TNF.Due to sequential homology in addition to functional homology, TNF was renamed as TNF-alpha, and LT was renamed as TNF-beta, they both belong to the TNF superfamily (Carswell et al., 1975). TNF is a proinflammatory cytokine secreted mainly by macrophages and involved in the regulation of cell proliferation, differentiation, and programmed cell death (Carswell et al., 1975;Männel et al., 1980;Nedwin et al., 1985).It also affects lipid metabolism, coagulation, and endothelial functioning (Pennica et al., 1984). Tumor necrosis factor-alpha (TNF-a) is an acute phase reactant cytokine produced by many cells, including granulocytes, T-lymphocytes, and neurons, but mainly by macrophages.Its gene is located within the major histocompatibility complex (MHC) region of chromosome 6 (6p21.33),with biased expressions in the bone marrow and lymph nodes.As a functional protein, TNF-a has two forms: bound (integral/transmembrane) and soluble.The soluble form is released from its original integral membrane or transmembrane (TP) form by a cleavage enzyme (metalloprotease) called (ADAm17) (Black et al., 1997). As an immune regulator, TNF-a is able to induce fever, cachexia, apoptosis, and inflammation.Additionally, it inhibits tumor genesis and viral replication.TNF binds to TNFR1, which is expressed by most cells, and activated by both the membrane-bound and soluble forms of TNF-a.However, TNFR2, which is primarily found in the immune system cells, is activated only by the membrane-bound form of TNF-a (Theiss et al., 2005). Protein signaling in the TNF-TNFR1 interaction occurs through the tumor necrosis factor receptor type 1-associated DEATH domain protein (TRADD), a 34 kDa adaptor protein that interacts with an intracellular domain (death domain) of TNFR1 after its dissociation from the inhibitory protein, SODD.The overexpression of TRADD is responsible for two major roles of TNF: anti-apoptosis and inflammation by activating the NF-ΚB pathway (Hsu et al., 1995).The inflammatory response of TNF is also induced by the inflammatory, pro-apoptotic MAPK pathway.The FADD/Caspase 8 pathway plays a minor role in the TNF induction of cell death (Chen and Goeddel, 2002;Lavrik et al., 2005). TNF is produced by the TNF gene cluster, which consists of three functional genes (TNFA, TNFB, and LTB) (Browning et al., 1993).Sequencing the entire coding region of the TNF-a gene shows the presence of five single nucleotide polymorphisms (SNP)-four in the upstream region and one in the non-translated region.In the upstream region, and with respect to the TNF transcriptional start site, there are three SNPs characterized by the substitution of adenine for guanine at nucleotides -238 (rs361525), -308 (rs1800629), and -376 (rs1800750) (Herrmann et al., 1998;Knight et al., 1999).The first two SNPs (rs361525 and rs1800629) are found in the promoter region, and they lead to increased TNF expression.These SNPs have been implicated in a variety of diseases, including autoimmune diseases, diabetes, coronary heart diseases, and cancer (Norman et al., 1995;Herrmann et al., 1998;Zinman et al., 1999).Additionally, the level of TNF-a is associated with poor prognosis and can expect treatment outcome in lymphoma patients (Salles et al., 1996).Among the most widely studied polymorphisms in TNF-a is -308G/A (rs1800629), in which the A allele is associated with an increase in transcriptional activity and the level of TNF-alpha.Reported studies from different institutions and geographic regions show no consistent relationships between -308G/A polymorphism and the risk of DLBCL.Both positive and negative associations have been reported between subjects of the same ethnicity and/or country of origin.For example, among American and Norway Caucasians, both Morton et al., (2008) and Yri et al., (2013) reported an increased risk of DLBCL with TNF -308G/A polymorphism (Morton et al., 2008;Yri et al., 2013).But Skibola et al., (2010) and Thunberg et al., (2010) found no such associations (Skibola et al., 2010;Thunberg et al., 2010).Among Asians, the results are inconsistent; some studies indicate that TNF-a G308A polymorphism is significantly associated with an increased risk of DLBCL while others show negative associations (Xiao and Zhang, 2011;Hosgood et al., 2013;Zhai et al., 2014).Our study among the Jordanian Arab population showed a positive association and an increased risk of DLBCL associated with TNF rs1800629G>A in the co- 11-4.31, 1.14-4.22, and 1.10-4.25, respectively; p value = 0.047, 0.014, and 0.016, respectively].The conflicting results of different studies indicate that the genetic effect of TNF-a 308G/A polymorphism on NHL risk is ethnically and geographically dependent and may be partially explained by different allele frequencies among different populations (6%, 5%, 7%, and 13% in Jordanian, South Asian, American, and European populations, respectively).Among our notable findings, the rs1799964T>C SNP in the TNF-a gene was associated with better overall survival (OS) in DLBCL patients.Kaplan-Meier analyses of overall survival rates revealed that patients with a dominant genotype (TT genotype in comparison with combined TC/CC genotypes) of TNF rs1799964 had a higher overall survival rate (log-rank p = 0.028). Lymphotoxin-A (LTA), or tumor necrosis factor beta (TNF-b), acts as a modulator in the immune system; it is a key regulator in lipid metabolism, secreted by lymphocytes, and has 35% identity and 50% homology in the amino acid sequence of TNF-alpha (Gray et al., 1984;Lo et al., 2007).The genetic location of LTA is (6p21.33)and induces its functions through the TNF alpha receptor-1 (TNFRSF1A/TNFR1) and TNF beta receptor-2 (TNFRSF1B / TNFR2) (Aggarwal et al., 1985). We found that lymphotoxin-a (LTA) 252A>G (rs909253) polymorphism was associated with an increased risk of DLBCL among our population (p = o.oo4), a result that is in concordance with previous reports in studies among the non-Hispanic white populations (Rothman et al., 2006). The tumor necrosis factor-alpha-induced protein 8 (TNFAIP8) is a protein with 188 amino acids and a molecular mass of 21 kD, located on chromosome 5q23.1 (Horrevoets et al., 1999).TNFAIP8 is an early anti-inflammatory cytokine and apoptosis regulator that negatively regulates T-cell receptor signaling, is involved in oncogenesis, and is found in many normal tissues and malignant cells.Its expression is induced by TNF-alpha through NF-KB pathway activation (Kumar et al., 2000).Head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, breast cancer, and non-small cell lung carcinoma show an increased expression of TNFAIP8 mRNA (Patel et al., 1997;Dong et al., 2010;Hadisaputri et al., 2012).However; the association between TNFAIP8 (rs1045241C>T) polymorphism and NHL, DLBCL in particular, is not well explored.In a study by Yan et al., (2012), the presence of TNFAIP8 (rs1045241C>T) polymorphism was significantly associated with the overall risk of DLBCL and follicular lymphoma (FL) among a Chinese population (Zhang et al., 2012).In our study, no significant relationship was found between TNFAIP8 rs1045241 C>T polymorphism and the risk of DLBCL. Leptin, or the obese (OB) gene, is a 16-kD endocrine (adipocyte-derived) protein mapped to chromosome 7 (7q32.1)and has an important role in regulating appetite, basal body metabolism, body weight, insulin level, and inflammatory response (Matsuda et al., 1997;Tian et al., 2002).Leptin mediates its effects through a cytokine single transmembrane domain receptor called leptin receptor (LEPR).Accordingly, leptin and LEPR are associated with the pathogenesis of obesity, diabetes mellitus (DM), blood pressure, and cancers (Skibolaet al., 2004;Lin et al., 2015;Zhang et al., 2018).Leptin gene expression is increased by sugar-rich diets and reduced by fasting.Polymorphisms in leptin (OB) and LEPR affect body weight homeostasis in different ways (Coleman, 1978;Thompson et al., 1997).LEP rs2167270 G>A polymorphism, which involves the untranslated 5-prime region of the gene and results in a single A-to-G transition in the 26th nucleotide, is associated with low leptin levels and morbid obesity (Hager et al., 1998).The obese phenotype is also associated with LEPR rs1137101 polymorphism, which results in the replacement of glutamine by arginine at codon 223 (Q223R (Gotoda et al., 1997).However, the relationship between body mass index (BMI) and the risk of cancers, including hematologic malignancies, is not well established, and previous studies show inconsistent results (Franceschi, et al., 1989;Wolk et al., 2001;Cerhan et al., 2002).In their studies, Skibola et al., (2004) and Willett et al., (2005) found that the presence of A allele associated with LEP rs2167270 G>A polymorphism decreases the risk of NHL, but both LEP rs7799039 G>A and LEPR rs1137101 (Q223R) polymorphisms are associated with increased risk of NHL (Skibola et al., 2004;Willett et al., 2005). These findings contradict the results of an Asian Chinese population study, in which no relationship was found between LEP rs2167270G>A and LEPR rs1327118C>G polymorphisms and risk of NHL (Zhang et al., 2012).Our results showed no significant association between LEPR rs1327118 G>C polymorphism and the patient's susceptibility to DLBCL.However, contrary to what was reported for the Chinese population, we found that LEP rs2167270 G>A polymorphism is associated with a decreased risk of DLBCL in the recessive mode models [odds ratio 0.47; 95% confidence interval (CI) 0.24-0.93;p = 0.032]. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "The study was supported by the Jordan University of Science and Technology Grant number 20170225 The study was approved by the Institutional Review Board of KAUH, Jordan.All the control subjects were voluntarily involved and signed a written informed consent form.All the clinical investigations were conducted according to the principles given in the Declaration of Helsinki. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "The study was supported by the Jordan University of Science and Technology Grant number 20170225 ",
"section_name": "Funding information",
"section_num": null
},
{
"section_content": "The study was approved by the Institutional Review Board of KAUH, Jordan.All the control subjects were voluntarily involved and signed a written informed consent form.All the clinical investigations were conducted according to the principles given in the Declaration of Helsinki. ",
"section_name": "Ethical approval",
"section_num": null
}
] |
10.3389/fncel.2018.00006
|
Rosuvastatin Improves Neurite Outgrowth of Cortical Neurons against Oxygen-Glucose Deprivation via Notch1-mediated Mitochondrial Biogenesis and Functional Improvement
|
Neurogenesis, especially neurite outgrowth is an essential element of neuroplasticity after cerebral ischemic injury. Mitochondria may supply ATP to power fundamental developmental processes including neuroplasticity. Although rosuvastatin (RSV) displays a potential protective effect against cerebral ischemia, it remains unknown whether it modulates mitochondrial biogenesis and function during neurite outgrowth. Here, the oxygen-glucose deprivation (OGD) model was used to induce ischemic injury. We demonstrate that RSV treatment significantly increases neurite outgrowth in cortical neurons after OGD-induced damage. Moreover, we show that RSV reduces the generation of reactive oxygen species (ROS), protects mitochondrial function, and elevates the ATP levels in cortical neurons injured by OGD. In addition, we found that, under these conditions, RSV treatment increases the mitochondrial DNA (mtDNA) content and the mRNA levels of mitochondrial transcription factor A (TFAM) and nuclear respiratory factor 1 (NRF-1). Furthermore, blocking Notch1, which is expressed in primary cortical neurons, reverses the RSV-dependent induction of mitochondrial biogenesis and function under OGD conditions. Collectively, these results suggest that RSV could restore neurite outgrowth in cortical neurons damaged by OGD in vitro, by preserving mitochondrial function and improving mitochondrial biogenesis, possibly through the Notch1 pathway.
|
[
{
"section_content": "Stroke is one of the leading causes of long-term disability and death worldwide and affects the patients' emotional, mental and physical health (Thampy and Pais, 2016).Neurogenesis including the regulation of neurite outgrowth is believed to be vital as a mechanism of neuroplasticity after cerebral ischemic injury (Kitamura et al., 2009;Lin and Sheng, 2015).Therefore, targeting neurite outgrowth represents a prospective therapeutic strategy for stroke patients. Neurite outgrowth is a developmental process that requires a heavy energy supply, provided by the mitochondria (Mattson and Partin, 1999).However, the disruption of oxygen and glucose supply, which is caused by stroke and mimicked by the in vitro oxygen-glucose deprivation (OGD) model, can produce a large number of reactive oxygen species (ROS) and lead to the depletion of cellular ATP (Rousset et al., 2015).Mitochondria are highly dynamic organelles and continually undergo biogenesis, fission and fusion (Anne Stetler et al., 2013).Maintaining a proper mitochondrial function depends on correct mitochondrial biogenesis (Sbert-Roig et al., 2016).Numerous studies have shown that mitochondrial dysfunction, especially regarding biogenesis, plays a crucial role in ischemic injury (McLeod et al., 2005;Gutsaeva et al., 2008).Therefore, identifying pharmacological agents that preserve mitochondrial functions and promote neurite outgrowth against cerebral ischemic injury might be an ideal therapeutic strategy. Statins are structural analogs of the 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA), the substrate of HMG-CoA reductase, and have been used as potent cholesterollowering drugs for the treatment of hypercholesterolaemia and coronary heart disease (Stein, 2002;Rader, 2003).Many studies have shown that statins reduce stroke incidence and improve its outcome (Bösel et al., 2005).The role of statins in neurite outgrowth has been proposed by previous studies (Jin et al., 2012;Métais et al., 2015).Rosuvastatin (RSV) is considered as one of the most effective statins and is able to ability to form multiple polar covalent bonds with the HMG-CoA reductase.In mice, RSV was shown to have a neuroprotective effect following cerebral ischemia.Accumulating evidence suggests that statins decrease the oxidative phosphorylation capacity and membrane potential of mitochondria, thus impairing their function (Broniarek and Jarmuszkiewicz, 2016), However, a recent study reported that, in the kidneys of wild type C57BL/6 male mice, RSV increases the protein levels of Sirt1 and PGC-1a, two key players in mitochondrial biogenesis (Corsetti et al., 2014).Nevertheless, it is not completely clear whether RSV modulates mitochondrial function and biogenesis during neurite outgrowth. The Notch pathway constitutes one of the most well-conserved developmental pathways throughout evolution.It controls both cell proliferation and apoptosis and is crucial for intercellular interactions in human development as well as in disease (Artavanis-Tsakonas et al., 1999;Bi and Kuang, 2015).A lot of attention has been paid to understanding how this pathway regulates cellular metabolism.Notch1, a well-studied protein, plays a significant role in this pathway (Kageyama et al., 2007).Recent studies have shown that the Notch1 pathway regulates mitochondrial fusion (Kasahara et al., 2013), but also affects their function (Basak et al., 2014), indicating that Notch1 is crucial in mitochondrial metabolism.Furthermore, the activation of Notch1 modulates the expression of important mitochondria-localized metabolic pathway proteins (Basak et al., 2014).Additionally, a previous study reported that Notch1 may exert a negative effect on neurite outgrowth (Berezovska et al., 1999).However, whether Notch1 mediates the protective effect of RSV on neurite outgrowth following ischemic injury is still not clear. Based on this evidence, this study was designed to investigate the role of mitochondrial function and biogenesis in RSV-induced neurite outgrowth.We also attempted to determine the role of Notch1 in promoting the effects of RSV, in order to further elucidate the potential mechanism of its action. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "The experimental protocols were conducted in accordance with guidelines approved by the Animal Experimentation Ethics Committee of Hebei Medical University. ",
"section_name": "MATERIALS AND METHODS",
"section_num": null
},
{
"section_content": "Cortical neurons were obtained from the brains of embryonic day 15-18 (E15-18) C57BL/6 mice (Vital River Laboratory Animal Technology Co. Ltd., Beijing, China).The cerebral cortex was dissected and incubated at 37 • C, for 15 min, in Hibernate-E solution (Sigma, Ronkonkoma, NY, USA), supplemented with papain (2.0 mg/ml, Sigma, Ronkonkoma, NY, USA).Then, the cortical tissues were neutralized and dissociated into single cells in Neurobasal medium containing 2% B-27 supplement (Invitrogen, Carlsbad, CA, USA) and 0.5 mM glutamine (Life Technologies, Carlsbad, CA, USA).Cells were plated at a density of 2 × 10 5 cells/cm 2 onto culture dishes, which had been coated with poly-L-lysine (Biocoat, BD Biosciences, San Jose, CA, USA), and grown in the same medium in a humidified 5% CO 2 incubator at 37 • C. In the present study, OGD was used to induce ischemia.To initiate ischemia, we used the same incubator in combination with a Hypoxic Workstation (gas mixture of 0.1% O 2 , 94.9% N 2 and 5% CO 2 , 37 • C). ",
"section_name": "Primary Cortical Neuron Culture and OGD",
"section_num": null
},
{
"section_content": "Neurons were pre-exposed to OGD conditions for 1 h and subsequently treated with different concertrations of RSV (0.5, 5, or 50 µM) for 48 h.Cells not treated with RSV or OGD served as a negative control.To evaluate the effect of Notch1 on mitochondrial function and biogenesis, a potent and specific inhibitor of the Notch1 pathway, DAPT, N-[N-(3,5difluorophenacetyl)-L-alanyl]-S-phenylglycinet-butyl ester, was added to the medium at 10 µM (in 0.1% dimethyl sulfoxide; Sigma, USA), 30 min before the RSV treatment. ",
"section_name": "Drug Application",
"section_num": null
},
{
"section_content": "After 48 h of drug application, primary cortical neurons were fixed with 4% paraformaldehyde for 20 min and then processed for immunocytochemistry.Briefly, neurons were incubated with the mouse monoclonal anti-β-III-tubulin antibody (Tuj-1, 1:500, Sigma, USA) overnight at 4 • C, followed by the donkey anti-mouse IgG, FITC-conjugated secondary antibody (1:200, CWBIO, China) for 1 h at 37 • C. Stained cells were imaged with an upright fluorescence microscope (Olympus, Japan).The length of the longest neurite of a Tuj-1-positive cell and the total neurite length per cell were measured using ImageJ software.Approximately 60 Tuj-1-positive cells per condition were measured. ",
"section_name": "Neurite Outgrowth Assay",
"section_num": null
},
{
"section_content": "The Mitochondrial Membrane Potential (MMP) of cortical neurons in different conditions was measured by using the JC-1 assay kit (Beyotime, China), according to the manufacturer's instructions.In brief, after the described treatments, neurons were collected and incubated with JC-1 staining solution (5 µg/mL) for 20 min at 37 • C. Cells were then rinsed twice with JC-1 staining buffer and centrifuged at 600× g at 4 • C for 15 min.The cells were resuspended with JC-1 staining buffer and the fluorescence intensity was detected using a monochromator microplate reader (Tecan, Switzerland).Fluorescence images were also obtained in green or red channels using an upright fluorescence microscope (Olympus, Japan).The fluorescence at 529 (green) and 590 (red) nm was measured using the monochromator microplate reader.The ratio of red to green fluorescence in different conditions was normalized to the respective one in the control condition, which was considered to have 100% MMP, and plotted graphically.Data were presented as percent of control. ",
"section_name": "Measurement of Mitochondrial Membrane Potential (MMP)",
"section_num": null
},
{
"section_content": "Intracellular ROS levels were quantified with the ROS assay kit (Beyotime, China) as previously reported (He et al., 2017).In brief, cortical neurons were incubated with 10 µM 2,7-dichlorofluorescein diacetate (DCF-DA) for 1 h at 37 • C in the dark and then resuspended in PBS.Intracellular ROS production indicated by the fluorescence intensity of the probe 2,7-dichlorodihydro-fluorescein diacetate (H 2 DCF-DA) was detected using a luminescence spectrometer with the excitation source set at 488 nm and the emission one at 525 nm.The values obtained at various conditions were expressed as the percentage change compared to the control condition. ",
"section_name": "Measurement of ROS",
"section_num": null
},
{
"section_content": "The cellular ATP levels were determined using an ATP assay kit according to the manufacturer's instructions (Beyotime, China).Luminesence was measured with a monochromatic microplate reader (Tecan, Switzerland).Data were presented as percentages compared to the control condition. ",
"section_name": "Detection of Cellular ATP Levels",
"section_num": null
},
{
"section_content": "Total DNA from cortical neurons was extracted using the DNeasy Blood and Tissue kit (Qiagen, Germantown, MD, USA) according to previous reports (Tian et al., 2017).Mitochondrial DNA (mtDNA) copy number was measured by real-time PCR using an ABI 7500 real-time PCR system (Applied Biosystems, Foster, CA, USA) with the SYBR Green detection method.The relative mtDNA copy number was determined by comparison to nuclear DNA (rRNA 18S).The primers for mtDNA were as follows: forward: 5 -AACACGA TCAGGCAACCAAA-3 , and reverse: 5 -GGTAGCGGGTGAGTTGTCAG-3 .The primers for rRNA 18S were: forward: 5 -GGACAGCGGGTGAGTTGTCA-3 , and reverse: 5 -ACCTTCGTTATCGGAATACC-3 . ",
"section_name": "Mitochondrial DNA (mtDNA) Quantification",
"section_num": null
},
{
"section_content": "Quantitative real-time PCR (qRT-PCR) was performed according to previous reports (Dai et al., 2014;He et al., 2016).Briefly, total RNA was isolated from cortical neurons using Trizol reagent (Invitrogen, Carlsbad, CA, USA).Reverse transcription was carried out using the First-strand cDNA synthesis kit (Fermentas International Inc., Burlington, Canada) and the cDNA was amplified by a real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) in the presence of a fluorescent dye (SYBR Green I, CWBIO).The relative abundance of specific mRNAs was calculated after normalization with the glyceraldehyde 3-phosphate dehydrogenase mRNA.The samples were tested in triplicates.Primers for all qRT-PCR experiments were listed as follows: NRF-1: forward: 5 -GAGTGACCCAAACCGAACA-3 , reverse: 5 -GGAGTTGA GTATGTCCGAGT-3 ; TFAM: forward: 5 -GGTGTATGAAGCGGATTT-3 , reverse: 5 -CTTTCTTCTTTAGGCGTTT-3 ; GAPDH: forward: 5 -AAGGTGAAGGTCGGAGTCAA-3 , reverse: 5 -AATGAAGGGGTCATTGATGG-3 . ",
"section_name": "Quantitative Real-Time PCR",
"section_num": null
},
{
"section_content": "Statistical analysis was performed using SPSS version 16.0.All data were presented as mean ± SEM.One-way analysis of variance (ANOVA) was performed for comparisons among groups, and SNK-q test was used for post hoc multiple comparisons.* p < 0.05 was considered to be statistically significant. ",
"section_name": "Statistical Analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "First, we examined the effect of RSV on the neurite outgrowth of cortical neurons under OGD.As shown in Figures 1A,B, neurons under OGD showed a remarkable decrease in neurite outgrowth (27.86 ± 5.11 µm, n = 60), compared to control cells (43.84 ± 7.15 µm, n = 60, p < 0.005).On the other hand, the treatment with different concentrations of RSV (0.5, 5, or 50 µM), for a period of 48 h after OGD injury, resulted in a significant recovery of neurite outgrowth.Compared with the OGD-treated cells (27.86 ± 5.11 µm), the length of the longest neurite was 30.25 ± 5.60 µm (n = 60, p = 0.016), for neurons treated with 0.5 µM RSV, 37.36 ± 6.55 µm, for those treated with 5 µM RSV (n = 60, p < 0.005), and 34.24 ± 7.02 µm, for those treated with 50 µM RSV (n = 60, p < 0.005).The fold change in the total neurite length between the untreated OGD-exposed cells and the treated ones with 0.5, 5 and 50 µM of RSV was 1.15, 1.65 and 1.44, respectively (n = 60, p < 0.005, Figure 1C).These results indicated that RSV could effectively improve neuritogenesis in cortical cells, previously injured by OGD.We chose 5 µM RSV to carry out subsequent experiments, considering that this concentration demonstrated the highest potential in inducing neurite outgrowth compared with the one of 0.5 (n = 60, p < 0.005) or 50 µM (n = 60, p < 0.005). ",
"section_name": "RSV Restores Neuritogenesis in Cortical Neurons Damaged by OGD",
"section_num": null
},
{
"section_content": "We next investigated the involvement of mitochondrial function in RSV-induced neurite outgrowth.Indicators of mitochondrial function were assessed in primary cortical neurons exposed to RSV under OGD conditions.Exposure of neurons to OGD for 1 h resulted in dissipation of the MMP (n = 6, p < 0.005) and an increase in ROS production (n = 6, p < 0.005).On the other hand, the treatment of cells with RSV, counteracted these effects. The MMP recovered (n = 6, p < 0.005; Figures 2A,B) and ROS accumulation was significantly reduced (n = 6, p < 0.005; Figure 2C), in comparison to untreated OGD-exposed cells. The above-mentioned results suggest that RSV can reverse the mitochondrial dysfunction, induced by OGD. ",
"section_name": "RSV Preserves Mitochondrial Function in Cortical Neurons under OGD",
"section_num": null
},
{
"section_content": "As mitochondria are the main source of energy generation, we detected ATP levels, to assess the energy metabolism of primary cultured neurons.RSV significantly reversed the decrease in cellular ATP levels, which was observed following exposure to OGD (n = 6, p < 0.005; Figure 2D).These results indicate that RSV can increase the energy metabolism, impaired by OGD. ",
"section_name": "RSV Elevates the Energy Metabolism of Primary Cultured Neurons Suppressed by OGD",
"section_num": null
},
{
"section_content": "To determine whether the altered mitochondrial function and energy metabolism are related to mitochondrial biogenesis, we also estimated the mtDNA content in different conditions (Figure 3A).The results showed that OGD exposure significantly decreased mtDNA content, whereas RSV treatment abrogated this effect (n = 6, p < 0.005; Figure 3A).Mitochondrial transcription factor A (TFAM) and nuclear respiratory factor 1 (NRF-1) are major regulators of mitochondrial biogenesis.Therefore, we measured the expression of these factors, using qRT-PCR (Figures 3B,C).The results showed that RSV treatment after OGD exposure significantly increased the mRNA expression of TFAM and NRF-1 (n = 6, p < 0.05; Figures 3B,C). ",
"section_name": "RSV Promotes Mitochondrial Biogenesis after OGD Exposure",
"section_num": null
},
{
"section_content": "Based on our finding that RSV preserved the mitochondrial function in primary cultured neurons exposed to OGD, we explored whether Notch1 was involved in this process.The release of the Notch intracellular domain (NICD) is widely used as a marker for Notch1 pathway activation.In our study, we examined NICD expression via immunocytochemical analysis.We found that NICD was expressed in cortical neurons, exposed to OGD, which suggested that endogenous Notch1 might be activated upon neuronal injury and mediate OGD remodeling.In these conditions, we found that RSV treatment increased the expression of NICD (Figure 4A; data not shown).Next, we used exposed to OGD.Compared to the RSV-treated cells, DAPT partialy decreased the mtDNA content (n = 6, p < 0.01; Figure 4E) and reduced the mRNA levels of TFAM (n = 6, p < 0.05; Figure 4F) and NRF-1 (n = 6, p < 0.01; Figure 4G).Additional, we did the following experiments whether DAPT itself had detrimental effects.The length of the longest neurite, total neurite outgrowth per cell, MMP, ROS, ATP, mtDNA, TFAM and NRF-1 were detected.The data were shown in the supplementary data.The results showed that there were no signifcant diferences in the length of the longest neurite, total neurite outgrowth per cell, MMP, ROS, ATP, mtDNA, TFAM and NRF-1 between OGD+DAPT and OGD group (see Supplementary Figures S1-S3 in Supplementary Data), suggesting that DAPT itself has no detrimental effects. ",
"section_name": "Notch1 Mediates the Protective Effect of RSV on Mitochondrial Function and Biogenesis in Cortical Neurons Exposed to OGD",
"section_num": null
},
{
"section_content": "In this present study, we demonstrated that RSV restores neuritogenesis in primary cortical cells damaged by OGD.This action is possibly mediated by the improvement in mitochondrial function and biogenesis.Furthermore, we also elucidated that Notch1 is crucial for these RSV-dependent effects.To the best of our knowledge, this is the first report to provide evidence for the effect of RSV on cortical neuritogenesis following OGD and the involvement of Notch1 in RSV-induced mitochondrial biogenesis and functional improvement. Statins act as inhibitors of the HMG-CoA reductase and have been extensively used for the treatment of hypercholesterolemia (Kahveci et al., 2014).Numerous studies have reported that statins may have neuroprotective properties, as demonstrated by the reduction of the affected region following focal cerebral ischemia and the protection of cortical neurons from excitotoxicity (Asahi et al., 2005;Bösel et al., 2005).Recent studies have shown that treatment of cortical neurons, cultured under OGD/reoxygenation conditions, with RSV, a novel HMG-CoA reductase inhibitor, was neuroprotective for the cells (Savoia et al., 2011).RSV exerts considerable protective effects on neural tissue against oxidative damage after spinal cord ischemia/reperfusion injury, improves cognitive functions in rats with diazepam-induced amnesia, and preserves long-term memory (Yavuz et al., 2013).Our results demonstrated that RSV, at a concentration of 5 µM, shows the maximum effects on enhancing neuritogenesis in cortical neurons exposed to OGD. Neurite outgrowth is an energy-consuming process that primarily depends on mitochondria.Mitochondria may play a crucial role in controlling neuroplasticity, including neurite extension (Mattson, 2007;Cheng et al., 2010).It has been reported that modulating mitochondrial function may impact neurite outgrowth (Habash et al., 2015).Furthermore, impaired mitochondrial function may disturb neuroplasticity following stroke (Cheng et al., 2010).Additionally, mitochondria may influnce the generation of ROS (Onyango et al., 2011), whose accumulation, caused by ischemia, could disrupt MMP and funcion.Damaged mitochondria can, in turn, generate more ROS (Bai et al., 2017).In this study, we found that the effects of RSV on neurite outgrowth correlated with improved mitochondrial function, as indicated by elevated MMP and ATP levels, as well as decreased ROS generation.These results suggest that mitochondrial function may be, at least in part, involved in RSV-induced neurite outgrowth. Mitochondrial biogenesis is a highly regulated process, which occurs continuously in healthy cells and is crucial for cellular adaptation (Nikoletopoulou and Tavernarakis, 2014).Recent evidence has suggested a subtle link between mitochondrial biogenesis and neurological disorders (Mandemakers et al., 2007).Mitochondrial biogenesis has been found to counteract the detrimental effects of oxidative stress and has been suggested as a novel target of the repair mechanism (Cheng et al., 2010;Habash et al., 2015).In vitro studies have also suggested that impaired biogenesis contributes to the reduction of mitochondrial function after cerebral ischemia (Wang et al., 2014); however, its enhancement may reduce ischemic brain injury (Valerio et al., 2011).Although previous studies have indicated that RSV might impair mitochondrial function and biogenesis (Broniarek and Jarmuszkiewicz, 2016), our results show that RSV treatment restores the OGD-induced mtDNA loss in cortical neurons.TFAM and NRF-1 play an important role in the initiation of mtDNA replication and the transcription of mitochondrial encoded genes (Campbell et al., 2012).Therefore, we measured the mRNA levels of TFAM and NRF-1 in neurons exposed to OGD and treated with or without RSV.Our results revealed that RSV treatment significantly increased the mRNA levels of these factors.Collectively, our findings indicate that the RSV-induced neurite outgrowth against OGD exposure can be partially explained by the improved mitochondrial function and their enhanced biogenesis.Recent studies have provided convincing evidence that RSV exerts its protective effect by decreasing ROS levels, inhibiting the opening of the mitochondrial permeability transition pore, and promoting mitochondrial biogenesis (Corsetti et al., 2014;Liu et al., 2017).Nonetheless, further studies will be required to determine the exact mechanism of RSV effects on mitochondria. The Notch pathway plays a vital role in the regulation of cell proliferation, self-renewal and differentiation, and is involved in several disorders of the central nervous system (Lundqvist et al., 2013).A previous study suggested that this pathway could regulate neurite outgrowth (Sestan et al., 1999;Levy et al., 2002).It also has been reported that Notch1 influences neurite morphology, and can activate its native signal transduction pathway in postmitotic neurons.Beyond neurogenesis, Notch1 plays a physiologically vital role in the central nervous system (Berezovska et al., 1999).In two recent studies, using M1 macrophages or cell lines in vitro, the Notch1 pathway was shown to enhance mtDNA transcription, ATP levels, and mitochondrial function (Basak et al., 2014;Xu et al., 2015).In addition, two statins, namely atorvastatin and simvastatin, have been shown to exert their effects, following stroke, through Notch signaling; the first, by increasing cell proliferation in the subventricular zone (Chen et al., 2008) and the second, by promoting arteriogenesis (Zacharek et al., 2009).Thus, we hypothesized that the Notch1 pathway was implicated in the regulation of RSV-induced mitochondrial biogenesis and function in cortical neurons.Our results show that, under OGD conditions, Notch1 signaling is active in primary cortical neurons and that its inhibition reverses the positive effects of RSV treatment on mitochondrial biogenesis and function.These results suggest that this pathway may, at least partially, contribute to the RSV-induced mitochondrial function and biogenesis in cortical neurons in vitro, which may represent a potent therapeutic strategy to promote brain plasticity after ischemic injury. ",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "The present study has demonstrated that RSV promotes neurite outgrowth in primary cortical neurons, thus shielding them against OGD.RSV seems to be vital in preserving mitochondrial function and improving mitochondrial biogenesis and these effects are, at least in part, mediated by the Notch1 pathway.These findings highlight Notch1 signaling and mitochondria as important players and novel therapeutic targets in promoting brain plasticity. ",
"section_name": "CONCLUSION",
"section_num": null
}
] |
[
{
"section_content": "WH and XT: study concept design and drafting of the manuscript.WH: collection of data.YL and XT: analysis and interpretation of data.All authors approved the final version of the manuscript. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fncel.2018.00006/full#supplementary-material FIGURE S1 | Effect of DAPT-only treatment on neurite outgrowth in primary cultured cortical neurons under oxygen-glucose deprivation (OGD).(A) Quantitative data of the longest neurite length of cultured cortical neurons.Results were presented as mean ± SEM. ns, non-significant, n = 60 per condition.(B) Quantitative data of total neurite length per cell.Results were presented as mean ± SEM. ns, non-significant, n = 60 per condition. ATP levels in cortical neurons exposed to OGD. (A) Mitochondrial membrane potential (MMP) was determined using JC-1 assay kit in different groups (OGD group, and DAPT group).Results were expressed as the mean ± SEM. ns, non-significant, n = 6 per condition.(B) The generation of ROS in different groups (OGD group and DAPT group).Results were expressed as the mean ± SEM. ns, non-significant, n = 6 per condition.(C) The ATP levels were measured in different groups (OGD group, and DAPT group).Results were expressed as the mean ± SEM. ns, non-significant, n = 6 per condition. The mitochondrial DNA (mtDNA) content was measured.Results were expressed as the mean ± SEM. ns, non-significant, n = 6 per group.The expression of mitochondrial transcription factor A (TFAM; B), and nuclear respiratory factor 1 (NRF-1; C) at mRNA levels was measured by Quantitative Real-Time PCR.Data are shown as mean ± SEM. ns, non-significant, n = 6 per condition. 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. Copyright © 2018 He, Liu and Tian.This is an open-access 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) or licensor 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": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "WH and XT: study concept design and drafting of the manuscript.WH: collection of data.YL and XT: analysis and interpretation of data.All authors approved the final version of 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/fncel.2018.00006/full#supplementary-material FIGURE S1 | Effect of DAPT-only treatment on neurite outgrowth in primary cultured cortical neurons under oxygen-glucose deprivation (OGD).(A) Quantitative data of the longest neurite length of cultured cortical neurons.Results were presented as mean ± SEM. ns, non-significant, n = 60 per condition.(B) Quantitative data of total neurite length per cell.Results were presented as mean ± SEM. ns, non-significant, n = 60 per condition. ",
"section_name": "SUPPLEMENTARY MATERIAL",
"section_num": null
},
{
"section_content": "ATP levels in cortical neurons exposed to OGD. (A) Mitochondrial membrane potential (MMP) was determined using JC-1 assay kit in different groups (OGD group, and DAPT group).Results were expressed as the mean ± SEM. ns, non-significant, n = 6 per condition.(B) The generation of ROS in different groups (OGD group and DAPT group).Results were expressed as the mean ± SEM. ns, non-significant, n = 6 per condition.(C) The ATP levels were measured in different groups (OGD group, and DAPT group).Results were expressed as the mean ± SEM. ns, non-significant, n = 6 per condition. ",
"section_name": "FIGURE S2 | Effect of DAPT-only treatment on mitochondrial function and",
"section_num": null
},
{
"section_content": "The mitochondrial DNA (mtDNA) content was measured.Results were expressed as the mean ± SEM. ns, non-significant, n = 6 per group.The expression of mitochondrial transcription factor A (TFAM; B), and nuclear respiratory factor 1 (NRF-1; C) at mRNA levels was measured by Quantitative Real-Time PCR.Data are shown as mean ± SEM. ns, non-significant, n = 6 per condition. ",
"section_name": "FIGURE S3 | Effect of DAPT-only treatment on mitochondrial biogenesis in cortical neurons exposed to OGD. (A)",
"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. Copyright © 2018 He, Liu and Tian.This is an open-access 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) or licensor 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": "Conflict of Interest Statement:",
"section_num": null
}
] |
10.1007/s00005-016-0433-7
|
The Expression of Toll-Like Receptors in Patients with B-Cell Chronic Lymphocytic Leukemia
|
B-cell chronic lymphocytic leukemia (B-CLL) presents with progressive accumulation of monoclonal B cells in the peripheral blood, bone marrow and lymphoid organs. B-CLL is characterized by heterogeneous clinical outcome. The expression of Toll-like receptors (TLRs) and their association with other prognostic factors in B-CLL patients remain unclear. The aim of our study was to evaluate the expression of TLR2, TLR4 and TLR9 genes and their significance as biological markers in patients with B-CLL. Sixty patients with newly diagnosed B-CLL were evaluated. The healthy control group included 20 age-matched individuals. Using quantitative reverse transcriptase PCR, the mRNA expression of genes TLR2, TLR4 and TLR9 was measured. TLR4 gene expression was lower in B-CLL patients as compared to the control group and TLR2 gene expression was higher in B-CLL patients than in healthy individuals. TLR9 gene expression was higher in the control group than in patients with B-CLL. TLR4 mRNA expression was lower in patients with advanced-stage CLL (Rai stages III and IV) than in patients with early stage disease (Rai stages 0-II). TLR2 gene expression was higher in patients with advanced-stage CLL (Rai stages III and IV) than in patients with early stage disease (Rai stages 0-II; p < 0.05). Our results suggest that TLRs could become potential biological markers for the clinical outcome in patients with B-CLL.
|
[
{
"section_content": "B-cell chronic lymphocytic leukemia (B-CLL) is the most common proliferative hematological disease in Western Europe and in the United States.B-CLL is associated with clonal proliferation of abnormal B lymphocytes, present in peripheral blood, bone marrow and lymphoid and extranodal organs (Chiorazzi et al. 2005).The pathogenesis of CLL is multifactorial and is associated with the activation of immune processes and with abnormal apoptosis (Caligaris-Cappio 2000).B-CLL has a variable clinical course and presentation.In some patients, the disease is chronic and does not require treatment for a long time, while in other patients the course of B-CLL is very aggressive.The negative prognostic factors in B-CLL include expression of ZAP70 and CD38, cytogenetic irregularities (deletion of 17p and/or 11q), and absence of somatic mutations in the immunoglobulin heavy chain variable (IGHV) genes (Zenz et al. 2010). Toll-like receptors (TLRs) are a crucial element of innate immunity.Up to date, 11 TLRs have been identified in the human body (TLR1-11) (Akira and Takeda 2004).TLRs appear on the surface of lymphocytes, monocytes, macrophages, and granulocytes.Through contact with the corresponding ligands, TLRs initiate a series of immune reactions which determine an anti-inflammatory response (Kawai and Akira 2007).TLRs are involved in the active processes connected with sepsis, autoimmune diseases, cancer as well as liver and cardiovascular diseases (Kawai and Akira 2011).Expression of TLRs on the surface of tumor cells may indicate their role in carcinogenesis and in their influence on tumor development and growth.Promotion of cancer is often proceeded by chronic inflammation which can also testify to the involvement of TLRs in malignant clonal expansion (Balkwill and Coussens 2004).It was also found that TLRs are involved in transferring cell signals through MAPK and PI3K cascades whose individual elements become disrupted during carcinogenesis (Hua and Hou 2013).The stimulation of TLRs plays a crucial role in the homeostasis of mature B cells; however, their participation in the etiology of CLL remains unknown.TLR2, TLR4 and TLR9 are involved in proliferation and differentiation of B lymphocytes. The aim of our study has been to evaluate expression of TLR2, TLR4 and TLR9, and their relevance as prognostic factors in patients with B-CLL. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "This study included 60 patients with B-CLL (29 women and 31 men).The median age of the patients was 68 (range 52-88).The examination protocol was approved by the Bioethical Commission.The diagnosis of B-CLL was consistent with the criteria IWCLL/NCI for CLL.40 patients (67%) were in the early stages of clinical progression according to the Rai staging system (0-II) and 20 patients (33%) were in stage III or IV according to the Rai staging system.All patients were previously untreated.The control group consisted of 20 healthy individuals (10 women and 10 men) in the age range similar to the studied population.Clinical data of the patients are shown in Table 1. Mononuclear cells of CLL patients and age-matched healthy donors were obtained from peripheral blood by density gradient centrifugation (Gradisol L, AquaMed, Ło ´dz ´) and stored at -75 °C in RNA isolation reagent (TriReagent Solution, Ambion/ThermoFisher) or as dry pellet. Total RNA was extracted from frozen cells by modified Chomczynski method according to the instruction delivered with TriReagent Solution and reverse-transcribed to cDNA with RNA-to-cDNA kit (ThermoFisher) (Chomczynski and Sacchi 1987). Relative expression of TLRs was assessed by real-time PCR using inventoried TaqMan Ò Assays from Life Technologies/ThermoFisher: Hs01152932_m1 for TLR2, Hs0015299_m1 for TLR4 and Hs0015973_m1 for TLR9.Beta-glucuronidase (GUSB) served as endogenous control (Hs99999908_m1).Reaction was performed in 7500 Real-Time PCR instrument (LifeTechnologies), using Gene Expression MasterMix (LifeTechnologies/ThermoFisher).Comparative C T method was used to compare the expression among patients and with healthy controls (Schmittgen and Livak 2008). The results were statistically analyzed using STATIS-TICA 8.0.Statistical analysis was performed by means of Mann-Whitney U test and p \\ 0.05 indicated a significant difference.Progression-free survival (PFS) was determined using Kaplan-Meier method.The long-rank test was used to compare the curves. ",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "In comparison to control group, TLR2 gene expression was higher in B-CLL patients than in healthy individuals (DCt TLR2 6.46 ± 9.58 vs 0.98 ± 0.43; p \\ 0.05).TLR4 gene expression was decreased in B-CLL patients in comparison to control group (DCt TLR4 2.21 ± 0.32 vs 11.91 ± 70.22; p \\ 0.05).TLR9 gene expression was higher in the control group than in patients with B-CLL (DCt TLR9 23.65 ± 16.29 vs 3.35 ± 1.93; p \\ 0.05). ",
"section_name": "TLRs Expression in Control Group",
"section_num": null
},
{
"section_content": "TLR2 gene expression was higher in patients with advanced-stage CLL (Rai stages III and IV) than in patients with early stage disease (Rai stages 0-II; p \\ 0.05).TLR4 gene expression was lower in advanced disease than in early stages (p \\ 0.05).The results are shown in Table 2. ",
"section_name": "TLRs Expression and Stage of CLL",
"section_num": null
},
{
"section_content": "We observed that expression of TLR2 in patients with anemia was significantly higher than in patients without anemia (DCt TLR2 17.18 ± 14.29 vs 7.10 ± 5.98; p \\ 0.05). We found significant positive relationships between TLR2 and white blood cells (r = 0.50, p 0.05), TLR2 and lactate dehydrogenase (r = 0.36, p \\ 0.05), TLR2 and beta2 microglobulin (r = 0.33, p \\ 0.05) and TLR4 and white blood cells (r = 0.29, p \\ 0.05).In addition, there was a statistically significant negative relationship between TLR2 and hemoglobin level (r = -0.43,p \\ 0.05). ",
"section_name": "TLRs Expression and Clinical Characteristics of Patients",
"section_num": null
},
{
"section_content": "We observed that in patients with positive ZAP70 expression, TLR4 expression was lower than in patients with negative expression (DCt TLR4 3.25 ± 0.52 vs 9.95 ± 3.22).This result was not confirmed by statistical analysis due to a limited number of cases.We showed no association between TLRs expression and CD38 expression. ",
"section_name": "TLRs Expression and ZAP70 and CD38 Expression",
"section_num": null
},
{
"section_content": "The median follow-up was 48 months (range 6-27).The disease progression occurred in 35 patients (58%).Patients with higher mRNA expression of TLR9 had significantly longer PFS in comparison to patients with lower mRNA expression of TLR9 (p \\ 0.05).The results are shown in Fig. 1. ",
"section_name": "Progression-Free Survival",
"section_num": null
},
{
"section_content": "Ten patients (17%) suffered from severe infections.Five patients had pneumonia and five patients had infectious diarrhea.Grade 4 infections appeared in patients with advanced disease.In these cases, TLR4 gene expression was decreased in comparison to patients with no or lower grade infections (DCt TLR4 4.26 ± 0.62 vs 10.15 ± 2.54).This result was not confirmed by statistical analysis. ",
"section_name": "TLRs Expression and Infections",
"section_num": null
},
{
"section_content": "B-CLL has a variable clinical course.The following prognostic factors are used in the clinical practice: expression of ZAP70 and CD38, cytogenetic abnormalities and absence of mutations in the IGHV genes whose presence allows the identification of the population of patients with B-CLL with a less favorable prognosis (Zenz et al. 2010). Despite this, prognostic markers that influence the course of B-CLL are still being searched for.Research has shown that TLRs are actively involved in the regulation of the differentiation and proliferation processes of B cells.In patients with B-CLL, they have shown a decreased gene expression of TLR2 and TLR4 and an increased gene expression of TLR7, TLR9 and TLR10 (Grandjenette et al. 2007;Muzio et al. 2009;Spaner et al. 2006).Previously, Barcellini et al. (2014) assessed the expression of the TLR4 and TLR9 genes in 95 patients with B-CLL.It was found that the expression of TLR4 is lower in the patient population than in the control groups.TLR9 gene expression was higher in patients than in healthy volunteers.Patients with B-CLL and with a decreased expression of TLR4 showed an increased risk of the disease progression and a higher incidence of autoimmune complications (Barcellini et al. 2014).The TLRs expression of genes in patients with B-CLL was also analyzed by Rozkova ´et al. (2010), who demonstrated a reduced gene expression of TLR-4 in patients with B-CLL.Gene expression figures for TLR1, TLR2, TLR6, TLR7 and TLR9 were similar to the control group (Rozkova ´et al. 2010).Arvaniti et al. (2011) marked the TLRs gene expression in a population of 192 patients with B-CLL.Their research confirmed a higher expression of TLR7 and a decreased expression of TLR2 and TLR4 in patients with B-CLL (Arvaniti et al. 2011). Our research on a population of 60 patients with B-CLL has demonstrated a decreased expression of the TLR4 gene compared with the healthy control group.TLR4 expression was also reduced in patients with advanced stages of B-CLL, as well as in patients with the expression of ZAP-70 and CD38, which are unfavorable prognostic factors in B-CLL.Our observations on the reduced expression of the TLR4 gene are analogous to the data available in the literature and show an influence of TLR4 on the clinical course of B-CLL.By analyzing the gene expression of TLR2, we have demonstrated a higher gene expression of TLR2 in the advanced stages of B-CLL.In the case of the TLR9 gene expression, our findings have proved inconsistent with the results presented in other studies.We have observed that the expression of the TLR9 gene was significantly higher in healthy subjects than in patients with B-CLL.The result of the impact of the TLR9 gene expression on PFS in the studied group is also interesting. In patients with B-CLL and higher, TLR9 gene expression correlated with significantly longer time relapsed before disease progression.This observation, however, requires confirmation in a larger group of patients with B-CLL.In patients with B-CLL, complications after serious infections often occur in connection with the impaired immune function of B cells.In the analyzed group of patients who experienced clinically significant infections, the TLR4 gene expression was reduced which may indicate the participation of TLRs in immune abnormalities in B-CLL. Our study shows a significant influence of TLR expression, and particularly of TLR4, on the clinical course of B-CLL.The decreased expression of TLR4 gene may suggest an unfavorable prognosis in patients with B-CLL.This observation, however, is preliminary and requires further research in a larger population of patients with B-CLL. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/),which permits unrestricted use, distribution, and reproduction in any medium, provided 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. ",
"section_name": "Discussion",
"section_num": null
}
] |
[] |
10.1371/journal.pgen.1005963
|
De Novo and Rare Variants at Multiple Loci Support the Oligogenic Origins of Atrioventricular Septal Heart Defects
|
Congenital heart disease (CHD) has a complex genetic etiology, and recent studies suggest that high penetrance de novo mutations may account for only a small fraction of disease. In a multi-institutional cohort surveyed by exome sequencing, combining analysis of 987 individuals (discovery cohort of 59 affected trios and 59 control trios, and a replication cohort of 100 affected singletons and 533 unaffected singletons) we observe variation at novel and known loci related to a specific cardiac malformation the atrioventricular septal defect (AVSD). In a primary analysis, by combining developmental coexpression networks with inheritance modeling, we identify a de novo mutation in the DNA binding domain of NR1D2 (p.R175W). We show that p.R175W changes the transcriptional activity of Nr1d2 using an in vitro transactivation model in HUVEC cells. Finally, we demonstrate previously unrecognized cardiovascular malformations in the Nr1d2tm1-Dgen knockout mouse. In secondary analyses we map genetic variation to protein-interaction networks suggesting a role for two collagen genes in AVSD, which we corroborate by burden testing in a second replication cohort of 100 AVSDs and 533 controls (p = 8.37e-08). Finally, we apply a rare-disease inheritance model to identify variation in genes previously associated with CHD (ZFPM2, NSD1, NOTCH1, VCAN, and MYH6), cardiac malformations in mouse models (ADAM17, CHRD, IFT140, PTPRJ, RYR1 and ATE1), and hypomorphic alleles of genes causing syndromic CHD (EHMT1, SRCAP, BBS2, NOTCH2, and KMT2D) in 14 of 59 trios, greatly exceeding variation in control trios without CHD (p = 9.60e-06). In total, 32% of trios carried at least one putatively disease-associated variant across 19 loci,suggesting that inherited and de novo variation across a heterogeneous group of loci may contribute to disease risk.
|
[
{
"section_content": "Congenital heart disease (CHD) is the most common congenital malformation and the most common cause of mortality during the first year of life in the United States [1,2].Most cases occur sporadically without a strong family history or identifiable genetic syndrome, and the primary heritable basis of most non-syndromic congenital heart disease has yet to be identified [3,4].Studies of affected kindreds and syndromic disease have revealed high-penetrance mutations at a small number of key loci [5].Exome sequencing and studies of structural variation of mixed cardiac phenotypes focusing on de novo events have identified novel disease loci in 4-10% of participants [6,7].However the remaining majority of non-syndromic subjects in exome and CNV studies are without an identified genetic cause. Atrioventricular septal defects (AVSD) are a rare cardiac malformation associated to date with a handful of canonical genes in cardiac development (NKX2-5, GATA4, GATA6, CRELD1) and may co-occur with certain rare syndromes.A recent study discovered causal variation in the nuclear receptor NR2F2 in 4% of 125 subjects with AVSD, pinpointing a single additional disease-associated gene [7].However the 4-10% discovery rate in studies of CHD highlight the observation that for any individual gene, highly penetrant de novo coding mutations may only account for a small portion of disease incidence, a phenomenon similar to the www.mouseatlas.org/.The RNAseq data from the second heart field and DMP generated by the Moskowitz lab has been submitted to GEO (GSE75077). Funding: JRP was supported by the Pediatric Scientist Development Program (NIH-NICHD K12-HD000850).The sequencing was supported by an in kind grant to EAA (NIH-NHLBI X01HL115206-01), and SAP corporation (Walldorf, Germany) which played no role in any portion of the study design, recruitment, data collection, or analysis.RPL is supported by Yale Center for Medelian Disorders (U54HG006504-04), and MB and DN are supported by the UW Center for Medelian Genomics (U54HG006493-04).IPM was supported by the NHLBI (R01 HL092153).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: EAA is a co-founder of Personalis Inc. which offers research and clinical sequencing studies similar to those described in this manuscript. sporadic occurrence and complex genetics of neurodevelopmental disorders [8].Therefore, expanding the scope of analysis in studies of CHD to include both inherited and de novo variation in multiple genes could increase the sensitivity of genetic studies of this heterogeneous group of oligogenic diseases [9][10][11][12][13]. To this end we assembled a multi-institutional cohort combining a discovery cohort of 59 trios with non-syndromic AVSD and a replication cohort of 100 single affected individuals and performed a genetic survey by exome sequencing and array-CGH.In a primary analysis we identified a novel candidate gene for AVSDs using inheritance modeling and prior knowledge of early cardiac gene expression (Fig 1).In secondary analyses, we searched protein interaction networks to identify the contribution of additional loci to this rare cardiovascular malformation.Finally we explored the contribution of rare inherited variation in genes related to other types of human and mouse cardiac malformations to AVSD. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "We determined the sensitivity of our informatics approach using a recently described consensus standard dataset of exonic 24,734 variants from NA12878 [14].Raw exome data from a well-characterized individual was analyzed with BWA/GATK 3.2 best practices and the RTG version 3.3.2software pipeline (Real Time Genomics Inc., Hamilton, New Zealand).RTG displayed greater sensitivity detecting 84.5% consensus standard variants compared to 79.9% for BWA/GATK (S1 Table ).Using the RTG pipeline we analyzed exome sequencing data on 159 individuals with AVSD but without a syndrome (a discovery cohort of 59 trios, and a replication cohort of 100 singletons derived largely from a published study of AVSDs (S2 Table ), along with 710 controls without congenital heart disease (59 trios, 533 singletons).The affected patients were situs solitus with a simple AVSD.Patients with other cardiac defects, heterotaxy, anatomical malformations, or developmental delay were excluded.Across all individuals, called variants displayed a median Ti/Tv ratio of 3.10, and a median of 89.6% phased genotypes within trio probands, which suggested an overall highly sensitive and accurate variant call set (S3 Table ).All de novo variants and insertions/deletions of interest were confirmed by Sanger sequencing. ",
"section_name": "The RTG Pipeline Yields a Highly Sensitive and Accurate Variant Call Set",
"section_num": null
},
{
"section_content": "Within the 59 AVSD trios and 59 control trios we analyzed variants with minor allele frequency of 0.03 or less in the 61,468 multiethnic individuals in the EXaC consortium [15].With the remaining protein-altering variants, we applied a rare-disease inheritance model to select only variants displaying classical inheritance patterns associated with sporadic presentation of a rare disease (de novo, homozygous, compound heterozygous) (Fig 1) [16]; this filtering process yielded a list of 710 variants in 399 genes in the 59 AVSD trios (S4 Table ). To identify novel genes involved in cardiac development and disease among the 399 loci, we reanalyzed 72 digital gene expression datasets derived from 22 tissues during mouse embryonic development (www.mouseatlas.org)[17] (S1 Fig) .The tissue types included the AV-canal along with 5 other cardiac tissues, and 16 other tissues from other organs or structures.After constructing co-expression modules using unsupervised weighted-gene coexpression network analysis, we observed that one of the co-expression modules expressed in mouse AV-canal tissue included four of six genes known to cause AVSDs (GATA4, GATA6, NKX2-5, and Analytical approach for disease gene discovery in a cohort of AVSD trios.Protein altering variants meeting allele frequency, quality, and read depth cutoffs from 59 trios were sorted with an inheritance model consistent with rare disease which combines de novo, compound heterozygous, and rare homozygous variants to collate variants into a list of genes [16].Three analytical approaches were applied to the final dataset.Primary Analysis.De novo variants in the AVSD trios were cross-referenced with the genes CRELD1, p = 6.57e-05, one-tail hypergeometric test) along with 69 of 756 genes related to other human or mouse cardiac malformations (S5 Table ) (p = 7.81e-08, one-tail hypergeometric test).These observations suggested the discovery of a co-expression module highly enriched for genes related to heart development and cardiac malformations. Intersecting the genes in this unique coexpression module (S6 Table ) with the 399 genes identified by the rare-disease inheritance model, two probands from the 59 AVSD trios displayed de novo mutations.One proband had a missense mutation in a non-conserved residue of KCNJ3 and a second proband had a missense variant in NR1D2.The KCNJ3 variant has been observed in low frequencies in European and African populations in the ExAC database and the available literature on Kcnj3 knockout animals did not suggest occult cardiovascular malformations [18,19].By contrast, the NR1D2 variant causes an arginine to tryptophan mutation at position 175 (p.R175W) in a highly conserved DNA binding domain (Fig 2a and2b).NR1D2 is a transcriptional co-repressor and modulator without a described role in cardiac development [20].De novo mutations in NR1D2 or any gene in the cardiac malformation module were absent from 59 control trios without congenital heart disease.Among the 61,468 putatively healthy individuals cataloged in the ExAC database there was only a single nonsynonymous mutation in the surrounding five protein residues surrounding the p.R175W allele.Overall the data were suggestive that this de novo allele might impact the function of NR1D2. ",
"section_name": "A Rare-Disease Inheritance Model and Developmental Coexpression Networks Identify NR1D2",
"section_num": null
},
{
"section_content": "To characterize the transcriptional activity of the p.R175W mutation we developed an in vitro transactivation assay performed in HUVEC cells, which employed a murine Nr1d2 expression vector co-transfected with an Nr1d2 response element (RE) consisting of 5 tandem repeats of a conserved binding site upstream of a minimal CMV promoter driving GFP.As NR1D2 is thought to act as a transcriptional co-repressor, a positive change in transcriptional activity of the RE vector may represent a decrease in the transcriptional co-repressor activity of Nr1d2.In this in vitro assay, the p.R175W mutation displayed an increased transcriptional activity relative to the wild-type allele (Fig 2c), which suggested that the p.R175W mutation in a conserved region of the DNA binding domain might functionally impair the native co-repressor function of NR1D2. The Nr1d2 tm1-Dgen -/-Mouse Shows AVSDs and Related Cardiovascular Malformations Though reports of a previous characterization of an Nr1d2 knockout allele did not show cardiovascular malformations [20], a percentage of homozygous knockout animals have been reported to die within hours of birth consistent with the presence of hemodynamically significant cardiac malformations [21].Two pairs of heterozygous founder animals were bred, yielding 17 pups (2 wild type, 7 Nr1d2 tm1-Dgen +/-, and 8 Nr1d2 tm1-Dgen -/-) which did not deviate obviously from expected Mendelian allelic ratios.Upon careful histological examination of two in a module highly enriched for CHD and cardiac development obtained from unsupervised weighted-gene coexpression network analysis to identify a novel AVSD gene.Secondary Analyses.Variants from the AVSD trios were mapped onto a protein interaction network followed by burden testing in a replication cohort (details in Fig 3).In a final analysis employing inheritance modeling, de novo, compound heterozygous, and rare homozygous loci observed in the AVSD trios were compared with a predefined list of genes associated with human or mouse cardiac malformations.Statistical comparisons were performed in a control group of 59 control trios without CHD.spontaneously deceased Nr1d2 tm1-Dgen -/-pups at P0 we detected a previously undescribed AVSD phenocopy (Fig 2d).We performed additional matings of +/-and -/-animals, and sacrificed mothers to obtain embryos at e16.5 and e17.5.A single -/-animal at e16.5 displayed an AVSD and a single -/-animal at e17.5 displayed an inlet ventricular septal defect which is closely related to AVSDs (Fig 2d).In total, 4 out of 15 -/-hearts assayed displayed a cardiac defect.Thus by combining inheritance modeling with a gene-coexpression network enriched for genes causing CHD, we identified a variant in NR1D2 which impacts transcriptional activity in vitro, and uncovered previously unrecognized cardiovascular malformations in an Nr1d2 knockout allele.Together these data suggest a new role for the transcriptional repressor NR1D2 in cardiac development and human disease.The p.R175W mutation shows increased transcriptional activity in a cell culture assay (p = 0.0022, two-tailed t-test).A wild-type or mutant Nr1d2 construct was transfected in HUVEC cells along with a vector containing a synthetic response element driving a GFP reporter.Mean and standard deviation for each condition are displayed.(d) Two representative cardiac lesions in Nr1d2 tm1-Dgen -/-mouse hearts.An oblique coronal section of the heart of a spontaneously deceased P0 Nr1d2 tm1-Dgen -/-pup reveals a primum ASD and suggests a common AV-valve (white arrow) indicative of AVSD (star).A coronal section of an e17.5 Nr1d2 tm1-Dgen -/-heart shows an inlet VSD (plus sign) which is a type of VSD closely related to AVSDs in cardiac development [78,83].Abbreviations: LV-left and RV-right ventricles, RA-right atrium, IVSinterventricular septum, VSD-ventricular septal defect, ASD-atrial septal defect.doi:10.1371/journal.pgen.1005963.g002 ",
"section_name": "The p.R175W Mutation Impacts Transcriptional Activity of Nr1d2 in vitro",
"section_num": null
},
{
"section_content": "The relatively low discovery rate in rare-variant association studies of CHD suggests that alternative analytical approaches may be necessary to distinguish the contribution of novel loci to disease risk [6,7].Protein interaction networks have successfully integrated known disease genes to discover the impact of novel loci in neurodevelopmental disorders and cancer, thus in a secondary analysis we employed an algorithm which searches protein-interaction data for over-representation of genetic variation within interacting proteins in the AVSD trios [22] (Fig 3). Including protein altering single nucleotide mutations and CNVs derived from the discovery cohort of 59 AVSD trios, the algorithm identified 86 enriched subnetworks of interacting An illustration of discovery of protein interaction networks for AVSD and validation by burden testing in separate a replication cohort.Filtered SNPs and CNVs from the AVSD trio probands are mapped to a protein interaction network (represented by grey dots and black lines), and the network is pruned to yield subnetworks (green dots and black lines).The subnetworks represent variants in genes which have verified interactions at the protein level, often constituting a portion of a signaling pathway or enzymatic complex [22].To validate the disease association of the individual subnetworks discovered in the trios, we performed burden testing for each subnetwork in a replication cohort of 100 AVSD cases originating largely from a separate study of AVSDs [10] along with 533 controls without congenital heart disease.doi:10.1371/journal.pgen.1005963.g003proteins containing 2 to 7 genes.By comparison, applying the algorithm to 59 control-trios identified 26 subnetworks of interacting proteins containing only 2 to 4 genes (Fig 4a).Using a procedure where the protein interaction network is randomly permuted, the genes within the AVSD-trio subnetworks were found to be enriched for true protein-protein interactions (median p = 0.01, network permutation procedure), while true protein-protein interactions were not observed within the control trio subnetworks (median p = 1.0, network permutation procedure) [23] (Fig 4a). To further characterize the protein interaction networks detected, we compared the subnetworks to gene expression in mouse cardiac development (S8 Table ).Genes within the AVSDtrio subnetworks were strongly overrepresented during mouse heart development (p = 9e-09, one-tailed hypergeometric test) while genes within the control-trio subnetworks were not (p = 0.34, one-tailed hypergeometric test) (Fig 4b).Thus, mapping genetic variation in the AVSD trios to protein interaction networks identifies 86 enriched subnetworks with deleterious variation in 231 genes that are preferentially expressed during cardiac development, a phenomenon not seen in the control trios without CHD. ",
"section_name": "Protein Interaction Networks Identified in the AVSD-trios are Expressed in Cardiac Development",
"section_num": null
},
{
"section_content": "To validate the genetic associations suggested by the discovered AVSD-trio subnetworks, we assembled a separate replication cohort of 100 singleton individuals (S2 Table ) and performed burden testing for each of the 86 protein networks (Fig 3).After Bonferroni correction for multiple hypothesis testing, a single subnetwork from the AVSD trios composed of a pair of interacting collagen genes (COL2A1, COL9A1) (Fig 4a and4c) displayed an elevated burden of rare coding variation in 100 affected individuals with AVSD compared to 533 controls without congenital heart disease (p = 8.37e-08, SKAT linear weighted test) (Fig 4c).Interestingly, the two genes COL2A1 and COL9A1 have evolutionarily conserved roles in cardiac development in both zebrafish and mouse [24][25][26].One mouse knockout allele of Col2a1 displays cardiac valve abnormalities [27], and mutations in both genes are associated with Stickler syndrome where 46% of patients are affected with congenital dysfunction of the mitral valve [28].Together the functional data on COL2A1 and COL9A1 accompanied by the identification of these genes with two separate methodologies in two separate cohorts of AVSD patients, support a potential association with other congenital structural malformations of cardiac valve tissue such as AVSDs. ",
"section_name": "Protein Interaction Networks Identified in the AVSD-trios are Validated by Burden Testing in a Replication Cohort",
"section_num": null
},
{
"section_content": "Outside of novel genes identified by developmental coexpression and protein interaction analyses, we wished to examine the impact of genes known to play a role in cardiovascular development or CHD in our cohort.Interestingly, de novo single nucleotide mutations in genes previously associated with AVSD (NKX2-5, GATA4, GATA6, EVC, CRELD1, NR2F2) were absent and we did not detect genes with recurrent de novo mutations. In an effort to categorize and catalog variation at known CHD loci within the 710 variants in 399 genes identified by the rare-disease inheritance model in the 59 AVSD-trios (S4 Table ), we assembled a predefined group of 756 loci associated with any human or mouse cardiac malformation (S5 Table ).Among the genes identified by the rare-disease inheritance model in the 59-AVSD probands (S4 Table ), we observed inherited and de novo variation in 16 genes (Table 1 andS7 Table) [29].Four of the affected probands displayed variation in more than one gene.In a set of 59 control trios without congenital heart disease we applied the identical variant calling pipeline and rare-disease inheritance model.Comparing the number of AVSD probands with inherited mutations in the identified 16 genes to unaffected controls, we observed 16 mutations in the AVSD-cases and only a single variant in controls (p = 9.60e-06, Fisher's exact test), and additional simulations confirmed an unusual distribution of mutations in the AVSD-cases compared to controls was unlikely to be a chance occurrence (p = 1.23e-06,Monte Carlo simulation).As an additional negative control we compared mutations in a list of 43 genes associated with congenital ocular malformations between the 59 AVSD cases and 59 controls; there was only a single inherited variant among the AVSD trios and none within the control trios (p = 1.0,Fisher's exact test).Together these results suggest a preponderance of de novo and inherited variation in genes associated with human or mouse cardiac malformations detected in the AVSD trios which greatly exceeded similar variation in control trios. Within the discovery cohort of 59 trios we also assessed structural variation by array CGH and read-depth analysis from exome studies.One patient was observed to have a 3.7 Mb de novo deletion at 8p23.1 encompassing 43 genes including GATA4 (Table 2a).An additional paternally inherited duplication at chr22:21,989,140-23,627,391 partially overlapping the congenital heart disease-associated 22q11.2duplication syndrome region was also detected [30]. Additional CNVs with a previous association to CHD were identified in the singleton subjects (Table 2b) [31].CNVs in these regions were absent from the trio and singleton controls.Thus, including inherited variants, de novo mutations, and structural variation, rare deleterious variants or CNVs in genes with strong prior evidence for association with congenital heart disease were observed in 14 of 59 or 23% of affected trios. ",
"section_name": "A Rare-Disease Inheritance Model Identifies Known CHD Loci in 23% of Trio Probands",
"section_num": null
},
{
"section_content": "In this study combining both exome-sequencing and array-CGH for a single specific cardiac malformation, we observed de novo and inherited variation in 19 genes associated with human disease, syndromic loci, and genes implicated in cardiac development by mouse knockout.In the absence of recurrent de novo events in a moderately sized cohort of 159 affected individuals, we applied an array of analytical techniques to look for both de novo and inherited variation associated with AVSDs. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "When combined with inheritance analysis, a gene-coexpression network derived from mouse development allowed us to identify a previously unrecognized role for the transcriptional repressor NR1D2 in cardiac development and human disease.Our experimental studies suggested that the observed p.R175W mutation impacts the transcriptional activity of murine Nr1d2, and we observed previously unrecognized cardiac malformations in the Nr1d2 tm1-Dgen knockout mouse.Yet within a cohort of 159 affected individuals, there was only a single patient with a de novo mutation in NR1D2, which highlights the underlying genetic heterogeneity of CHD and the utility of applying orthogonal datasets to pinpoint causal variation.The -/-animals for the Nr1d2 tm1-Dgen allele display incomplete penetrance; a majority of animals do not display cardiovascular malformations and develop normally to adulthood displaying phenotypes related to circadian rhythm and abnormal lipid metabolism [20].Nr1d2 may retain multiple roles in modulating transcription but is most clearly described as a transcriptional repressor, therefore a mutation in the DNA binding domain might impact Nr1d2 to binding to target sequences resulting in a \"de-repression\" transcriptional targets of Nr1d2.A knockout allele such as Nr1d2 tm1-Dgen might similarly \"de-repress\" targets of Nr1d2.From the standpoint of transcriptional repression, increased transcription of the p.R175W mutant observed in vitro may represent a decrease in transcriptional repression relative to the wildtype protein, and as such could be entirely consistent with the phenotype of a mouse knockout allele.Interestingly, NR1D2 is a well-characterized component of the molecular clock, and further studies would be necessary to investigate NR1D2 as a link between the molecular clock and timing of cardiac development. Chromatin remodeling factors have recently been implicated as primary and secondary causal factors in CHD [6,32], and both newly discovered factors NR1D2 and NR2F2 may play integrated roles in chromatin remodeling during cardiac development.The key histone deacetylase HDAC1 is directly activated by NR1D2 binding and indirectly activated by NR2F2 via PROX1 [33][34][35].Additionally NR1D2 may function upstream of NR2F2, modulating the autoregulatory activity of NR2F2 via HDAC1 and the glucocorticoid receptor GR complex [36,37].Further experiments are necessary to delineate the tissue localization, timing, expression, and functional roles of these two transcription factors and their role in chromatin modulation and transcriptional regulation during cardiac development. ",
"section_name": "Coexpression Networks Suggest a Novel CHD Locus",
"section_num": null
},
{
"section_content": "Within a complex cellular or tissue signaling pathway, capturing the genetic variation in one or more interacting proteins has yielded novel candidate genes in cancer and neurodevelopmental disorders [22,38].Adapting a tool designed to search protein interaction networks in cancer, we identified a small number of variants in genes within the AVSD trios, two of which (COL2A1 and COL9A1) were subsequently validated in burden testing of a separate replication cohort of 100 individuals at a statistically significant threshold.Independent experimental data implicates these genes in the development of the cardiac valve structures, and links these genes to a genetic syndrome that includes abnormalities of the cardiac valves among a host of other phenotypes.Though our observations are not firmly conclusive of a causal role for COL2A1 and COL9A1 in the pathogenesis of AVSDs, they are supportive of such a role, and we believe, consistent with the idea that network-based approaches may be fruitfully applied to gene discovery in CHD phenotypes. ",
"section_name": "Exploring Oligogenic Inheritance with Protein Interaction Networks",
"section_num": null
},
{
"section_content": "Surprisingly, with the exception of a deletion encompassing GATA4 seen in one trio subject and one singleton subject (Table 2), we did not discover de novo coding mutations or gene dosage alterations within the 59 trios in canonical AVSD genes (NKX2-5, EVC, CRELD1, GATA6) or at the newly discovered CHD risk locus NR2F2.This finding is consistent with studies of CHD examining candidate genes [39] and exome sequencing where protein-altering variants in any single gene are reported in no more than 1-4% of patients [7].The absence of recurrent de novo variants in a cohort of 59 patients with AVSD is in striking contrast with other cardiac conditions such as long QT syndrome where pathogenic coding variation in only 5 genes accounts for disease in 70% patients [40].We hypothesize that the absence of de novo variation observed at canonical loci in a cohort of this size reflects the complexity of CHD genetics and highlights the utility of considering alternative inheritance patterns to detect disease-associated variation. ",
"section_name": "Absence of Recurrent Mutations in Canonical AVSD Genes",
"section_num": null
},
{
"section_content": "Among a list of 756 genes with either a clinical or experimental association to cardiac malformations (S5 Table ) we observed rare inherited variants in the AVSD trios that were not seen in control trios [29].In genes clinically associated with CHD we observed compound heterozygous variants inherited in trans in ZFPM2, NSD1, NOTCH1, VCAN, and MYH6 and rare homozygous variants in MYH6.Variation was observed in 9 additional genes including compound heterozygous variants in ADAM17, CHRD, IFT140, PTPRJ, and RYR1 and rare homozygous variants in ATE1, and the presence of heart defects in mouse knockout models for these loci supports their association with human cardiac malformations. In an independent forward mutagenesis screen Ift140 causes AVSD among a variety of congenital malformations [41].The calcium channel RYR1 is associated with skeletal myopathies and malignant hyperthermia, but primum atrial septal defects in one mouse allele suggest a role in early cardiac development [42].The metalloproteinase ADAM17 may link NOTCH1 signaling in cardiac valve development to the left-right patterning of the heart [43,44].Individual knockout alleles of CHRD, PTPRJ, and ATE1 each show defects in heart development recapitulating different human malformations [45][46][47].Importantly, we observed rare inherited variation in genes with experimental or clinical evidence for a role in cardiac development and CHD within the AVSD trios, but rare inherited variation in these same genes was largely absent from the control-trios. ",
"section_name": "Rare Inheritance Models Identify Genes Associated with CHD and Cardiac Development",
"section_num": null
},
{
"section_content": "Despite excluding syndromic features and developmental delay from our patients at the time of recruitment, we observed inherited and de novo variation in genes causing syndromic disease that include heart malformations.A de novo mutation was detected in an unknown protein domain of EHMT1 the causal gene in Kleefstra syndrome, compound heterozygous variants inherited in trans were observed in SRCAP which was recently associated with Floating-Harbor syndrome, two individuals showed compound heterozygous mutations in BBS2 which causes Bardet-Biedel syndrome, a fifth individual displayed compound heterozygous mutations in NOTCH2 which causes Alagille syndrome, and a sixth individual displayed a compound heterozygous mutation in KMT2D the locus implicated in Kabuki syndrome.Each of these multi-organ syndromes is frequently accompanied by AVSD or another related form of congenital heart disease [48][49][50][51][52]. Within a single locus associated with a genetic syndrome, different alleles may vary in their expressivity.We hypothesize that these variants represent hypomorphic alleles of syndromic genes, where the patients affected present only one aspect of the phenotype associated with the syndrome, in this case a phenocopy of a syndromic associated cardiovascular malformation [53].Indeed on secondary followup, none of the included probands with EHMT1, KMT2D, SRCAP, or NOTCH2 variants displayed other characteristics of their associated syndromes, while the patients with BBS2 mutations were not available for review (additional phenotypic information on patients carrying syndromic alleles is detailed in the S1 Text).Supporting the possibility of hypomorphic alleles, there was a striking absence of de novo or inherited variants in these syndromic genes within the control trios suggestive that the discovered variants may confer risk for AVSD. ",
"section_name": "Syndromic Alleles in Nonsyndromic Patients",
"section_num": null
},
{
"section_content": "These findings have limitations.Although we excluded patients with a family history of cardiac malformations, in an earlier era of surgical care the parents of the study participants would have been less likely to survive to reproductive age [54].In our study the parents received only a questionnaire and did not receive screening echocardiogram, thus we cannot rule out that a parent in an included trio may have a forme fruste of an AVSD-related malformation such as a cleft mitral valve or ostium primum ASD.Additionally, there is emerging evidence that maternal risk factors (both genetic and environmental) which confer risk for CHD that were not considered in our study design [55][56][57]. Genetic studies of CHD are challenged by the fact that specific individual malformations are quite rare (AVSD is approximately 0.3 per 10,000 live births), and that any substantial group of patients with a single malformation will contain some population stratification.The unexplored role of non-coding gene regulatory variation in congenital heart disease is not surveyed by our exome-sequencing approach [58,59].The power of SKAT tests are likely limited by a small cohort size, the heterogeneous genetic backgrounds of the case and control populations, the differences in exome sequence capture and sequencing chemistries employed, the absence of an inheritance model, and most importantly the underlying complex oligogenic architecture of cardiac malformations [12,60,61].Finally there are no statistical models that account for ethnicity in models of rare-variant transmission, therefore the influence of population stratification or ethnicity upon our rare-inheritance model of disease is not known. Overall our analysis suggests locus heterogeneity in the pathophysiology of a single cardiac developmental malformation.We observed recurrent variation within three genes (GATA4, MYH6, and BBS2) in only 6 of 59 trios.Including inherited, de novo, and discovered loci, 32% of trios displayed one or more putatively contributory mutations in the 19 genes identified.Supported by both experimental and clinical evidence, we suggest that inherited rare variants with a moderate effect size across multiple loci may impact the risk of congenital heart disease in addition to de novo variation.Taken together our catalog of 19 loci with experimental evidence for disease among 159 patients is consistent with the long-hypothesized oligogenic inheritance of congenital heart disease [12]. ",
"section_name": "Limitations and Conclusions",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": null
},
{
"section_content": "The guidelines of the Declaration of Helsinki were followed and the study was approved by the institutional review board of Stanford University (IRB-23637, IRB-23572) along with each institution from which participants were recruited.Written informed consent was obtained from each participant.Trios or single patients with an AVSD and situs solitus were recruited.We excluded patients with other major congenital malformations, developmental delay, or other types of CHD (excluding patent ductus arteriosus or secundum atrial septal defect).All participants were assessed clinically by pediatric cardiologists or clinical geneticists to exclude other syndromes associated with AVSD or CHD.Participants were obtained from Seattle Children's Hospital [62], the Pediatric Cardiac Genomics Consortium, the University of Iowa, and reanalyzed from a published study of AVSDs drawn from patients at the University of Toronto [7] (S2 Table ). ",
"section_name": "Ethics Statement, Case Description, and Patient Cohorts",
"section_num": null
},
{
"section_content": "Exome sequences for 531 control subjects of Caucasian and African American descent without CHD were derived from the atherosclerosis risk in communities (ARIC) consortium warehoused at dbGAP, and from local recruitment efforts.An additional 59 control trios (healthy parents with a healthy child) were obtained from the Simons Foundation.Raw sequence data in the form of bam or fastq files was re-aligned and re-analyzed with the below-described bioinformatic pipelines. ",
"section_name": "Control Cohorts",
"section_num": null
},
{
"section_content": "DNA was isolated by standard techniques from either whole blood, saliva samples, or immortalized lymphoblasts.Exome sequencing was performed for all complete trios and single affected individuals at two academic centers (University of Washington or Yale University) and two commercial sequencing providers with the SeqCap EZ Human Exome Library v2.0 (Roche NimbleGen, Madison, Wisconsin, USA), SureSelectXT Human All Exon V4 (Agilent Technologies Inc., Santa Clara, California, USA), or a proprietary capture library based on the Agilent SureSelectXT Human All Exon V5 platform (Personalis, Corp, Menlo Park, California, USA).Paired-end sequencing was performed on Illumina HiSeq 2500 machines with 75-, 100-, or 150-bp read lengths in all but two trios, which were sequenced with 33 bp paired end reads.For all included samples Median Ts/Tv was 3.10 while coverage depth was 43.8x (S3 Table ).Exome sequencing on single individuals from the Toronto cohort was performed as described [7], and for subset of unrelated individuals raw fastq files were obtained and reanalyzed via the below described bioinformatics platform for the purposes of reanalysis. AVSD trios and the singleton/replication patients not originating from Toronto were assayed for CNVs by array comparative genomic hybridization (CGH) by using a custom chip described previously or the SurePrint G3 Human CGH Microarray Kit, 8x60K (Agilent Technologies Inc., Santa Clara, California, USA) with described protocols [31,62].When a CNV was detected in an affected participant, a dye-swap experiment was repeated to ensure reproducibility and the parents were then assayed if available to determine inheritance status.For detection of smaller variation, two exome CNV detection assays using read depth data were also employed for all participants [63,64].When there was agreement in a structural variant call between at least two calling methods for any variant of interest, we employed an orthogonal CNV genotyping assay when DNA was available (for all of the AVSD trios and the singleton/ replication patients not originating from Toronto).The CNV genotyping assay was carried out with 10 ng of DNA per manufacturer instructions against the RNaseP CNV reference assay (Life Technologies, Carlsbad, California, USA).The assay was run on a ViiA 7 Real Time PCR System (Life Technologies, Carlsbad, California, USA) for 40 cycles under standard reaction conditions, and CNV genotypes were called with the copycaller software [65]. ",
"section_name": "Exome Sequencing and CNV Detection",
"section_num": null
},
{
"section_content": "Two genotyping pipelines were tested.A rapid and sensitive commercial software package, rtgcore version 3.3.2was applied to the raw exome sequence data for mapping, pedigree-aware variant calling, and genotype filtration (Real Time Genomics Inc., Hamilton, New Zealand) [66] to the UC Santa Cruz human genome reference sequence (hg19) (S1 Script).A second pipeline based on the HugeSeq BWA/GATK HaplotypeCaller pipeline was also employed for purposes of comparison. To determine the accuracy of our genotyping pipeline we re-genotyped available exome data from NA12878 for comparison to a recently described gold standard dataset of 24,734 variants from this individual [14].This set of variants from the consensus standard dataset was limited to the exome capture region of the nextera kit all human exome v2 (Illumina Corp, San Diego, California, USA) and regions of suspicious variant quality [67] were excluded to yield a true positive dataset of 24,734 true positive variants from NA12878.The vcfeval tool from RTG was used for all comparisons of vcf files as it robustly handles the different possible textual representations of insertions, deletions, and substitutions that may be produced by different genotyping algorithms.Using raw fastq files generated by the Garvan Institute, comparing the unfiltered output of both pipelines the two algorithms both called 19,566 variants (79.9%) of the NA12878 true positive dataset (TP) in common.RTG called an additional 1,469 TP, compared to BWA/GATK which only called an additional 198 unique TP variants.Overall RTG displayed a greater unfiltered sensitivity at 84.5% compared to 79.9% for BWA/GATK; therefore we selected the RTG pipeline for further analysis of the cohort (S1 Table ). To classify variants we selected an AVR score of 0.5 to balance a sensitivity of 99.4% and positive predictive value of 90.3% for the purposes of variant discovery.Alignments for all variants of interest were manually inspected with the IGV software, and all de novo coding variants and any insertion/deletion of interest was confirmed by direct Sanger sequencing of PCR amplicons, or alternately clonal Sanger sequencing of 12 colonies from cloned PCR amplicons.For burden testing, pooled simultaneous variant calling for all included cases and controls was performed with the RTG population caller and variants filtered for a read depth of 8 and AVR score of 0.5.Analyses were limited to the regions of intersection of the bed files of the exome capture kits obtained from the respective manufacturers.Known artifactual variants arising from exome sequencing were removed at the time of variant filtration [67,68]. ",
"section_name": "Genotyping Pipeline",
"section_num": null
},
{
"section_content": "All statistical analyses employed the R language for statistical computing version 3.1 unless otherwise specified.For population analyses we selected 8,940 snps from the Affymetrix Genome-Wide Human SNP Array 6.0 (Affymetrix, Santa Clara, California, USA) common to the five exome capture protocols employed in the study.All included probands were re-genotyped for this limited set of variants, and combined with individual level data from 1032 diverse samples of known ethnicity from the 1000 genomes project.The MDS and kinship modules from the KING software were used to estimate ethnic background and five individuals displaying cryptic familial relationships from the Toronto cohort among the singletons were identified and excluded from further analysis [69].A multinomial linear model was built for each population and the presence of admixture from the populations of the 1032 known samples, and used to infer ethnic background and the presence of admixture in the included probands.Selfreported ethnicity was available for 547 individuals, which was 97.6% concordant with predicted ethnicity. Protein altering variants were sorted for minor allele frequency less than 0.03 and prioritized by inheritance patterns consistent with rare disease (de novo, rare homozygous, and compound heterozygous) using the trioTools module from the STMP package [16].Protein-altering variants from the 59 trios were selected, haplotypes constructed, and variants phased with the PLINKseq software package [70].Imputation was not performed thus all variants analyzed originated from the primary genotyping pipeline. After sorting for inheritance consistent with rare disease (de novo, rare homozygous, and compound heterozygous), variants were collated and assembled into lists.A blacklist of genes with low prior likelihood of causality was excluded from further analysis, which included genes with a residual variation intolerance score greater than 90 and genes with copy number polymorphisms [68,71,72]. For statistical comparisons between the groups of trios, the number of individuals with rare inheritance (de novo, rare homozygous, or compound heterozygous) in these genes in the 59 trios and 59 controls was counted and compared with a Fisher's Exact test.As the underlying distribution combining de novo, rare homozygous, and compound heterozygous inheritance into a unified \"rare inheritance\" model is not readily estimated from available genotyping data, and because variant detection (particularly de novo variation) may vary systematically with the technical aspects of sequencing (variant calling algorithm, read depth, exome capture platform, and sequencing chemistry) we estimated empiric p-values by Monte Carlo simulation.To simulate an underlying distribution we performed permutations drawing 16 genes randomly from a list of 18,495 protein-coding genes, the rare inheritance events for each random list counted within 59 cases and 59 controls by individual, and a Fisher's exact test applied. The p-value was estimated with the formula (r+1)/(n+1) where n is the number of simulated replicate samples and r is the number of test statistics exceeding the calculated test statistic from the observed data (p = 9.60e-06).This simulation procedure suggested an empiric p-value of 1.23e-06, a similar order of magnitude as the p-value calculated from the observed data. Gene modules or subnetworks identified by protein interaction networks in the HotNet2 algorithm (see below), were subjected to burden testing with the SKAT linear weight test and a Bonferroni correction for multiple hypothesis testing employed [73,74].The first four principal components from a principal components analysis of all variants were used as covariates for burden testing.Within the SKAT algorithm variant weighting was derived from a beta density function (p i , 1, 25) where p i is the minor allele frequency.For variant weighting, minor allele frequencies were derived from the 61,428 individuals in the multiethnic EXaC dataset (version 0.2 http://exac.broadinstitute.org/),while for variants not observed or reported in the EXaC dataset minor allele frequencies were calculated from frequency observed among the genotyped individuals.The quantile-quantile plots for the SKAT linear weight test of 86 subnetworks (inclusive of combinations of 231 single genes-see below) suggest that the test-statistics derived are controlled for ethnicity or other systematic differences between cases and controls such as coverage (S2 Fig). ",
"section_name": "Statistical Analysis",
"section_num": null
},
{
"section_content": "Unique tag-count data from 74 SAGE libraries representing 22 tissues constructed as a part of the mouse atlas of gene expression project were downloaded from www.mouseatlas.org[17].SAGE tags were mapped with the Burrows-Wheeler aligner to the 115,746 unique mouse RefSeq transcripts downloaded from UC Santa Cruz website (http://genome.ucsc.edu)and tag data converted to a digital gene expression format constituting a tag counts per transcript using custom Perl scripts.Tags matching pseudogenes were removed.Using the R environment for statistical computing, tag counts by transcript were normalized by library size with the EdgeR package and collapsed to gene by connectivity with the WGCNA package [75,76].A standard WGCNA workflow for digital gene expression was applied to the normalized and collapsed data for coexpression module construction followed by correlation to the tissue of origin and annotation with the human orthologous gene name when available (S2 Script).To independently assess the predictive capacities of our unsupervised network building procedure, we observed that the developmental expression of 13 well characterized congenital heart disease genes are accurately localized by the network model to their appropriate cardiac tissue, suggesting excellent specificity for detecting developmental cardiac related gene expression (S9 Table ).Comparison of variant and gene lists for under-and over-representation were performed with a one-tailed hypergeometric test.Gene expression networks were visualized with the Gephi software package [77].Because the DMP is a key structure in development of the atrioventricular septum [78] and was not explicitly included in the original set of micro-dissected tissues from the mouse atlas of organ development (www.mouseatlas.org),we added the DMP gene expression data to the cardiac development gene expression set.Among genes expressed in the DMP we selected the most highly expressed 1000 genes across 6 datasets generated from the posterior second heart field including the dorsal mesenchymal protrusion.The microdissection of the posterior heart field was performed in the laboratory of Dr. Moskowitz at embryonic mouse tissue at E9.5, subject to reverse-transcription, amplification, and sequencing by The University of Chicago Genomics Core (GSE75077). ",
"section_name": "Co-expression Network Analysis from Mouse Organ Development",
"section_num": null
},
{
"section_content": "Variant data annotated with the STMP package [16] including SNVs and CNVs from the 59 AVSD trio probands and 59 control trio probands was converted and formatted with custom python scripts, and included all protein altering variants with a minor allele frequency in EXaC of 0.02 or less were included in the analysis using four included protein interaction networks.For each of three protein interaction networks (Multinet, IrefIndex9, and HINT [22]) four delta values were derived with the network permutation procedure and applied to identify subnetworks.The resulting subnetworks identified by the HotNet2 algorithm were manually inspected for validity, and the subnetworks from the MultiNet protein interaction networks were chosen for further analysis.For each set of subnetwork sizes ranging from 2 to 10, the HotNet2 algorithm derives a p-value from the hypergeometric distribution comparing the observed number of subnetworks of size n within in a dataset compared to an expected number of subnetworks.The expected number of subnetworks is derived from a computationally intensive network permutation procedure; in this case the MultiNet protein interaction network was subject to 100 permutations limiting the range of p-values to a minimum p-value to 0.01 and maximum p-value to 1.For the AVSD-trio subnetworks the median p-value was 0.01 across the nine subnetwork sizes and four derived delta values, in comparison to the control-trio subnetworks where the median p-value was 1.0; this suggested an enrichment in variants occurring in genes with true protein-protein interactions within the AVSD-trios (rather than randomly occurring simulated protein-protein interactions in the permuted networks).A single output run utilizing the MultiNet protein interaction network applying a delta value of 0.00126036397514 yielded 86 subnetworks containing 231 genes in the AVSD trios, and the 86 subnetworks were subject to burden testing in the singleton cohort (see above).Protein interaction data were processed with custom python and shell scripts for visualization using the Gephi software tool [77]. ",
"section_name": "Protein Interaction Networks with HotNet2",
"section_num": null
},
{
"section_content": "Rare inherited variants in the AVSD-trios.The variant calling and filtering process in the 59 AVSD-trios produced a list 710 variants in 399 genes (S4 Table ). The AVSD Coexpression Module.The coexpression module highly expressed in the AV-Canal contains 934 genes including four of six known genes causing AVSDs, including NKX2-5, GATA4, GATA6, and CRELD1 (S6 Table ). Known CHD genes in human & mouse.To develop a list of 756 genes related to cardiovascular malformations in humans an mice we combined a list of 85 genes previously associated with human cardiac malformations [29] along with a list of 671 mouse genes known to cause cardiac malformations similar to human disease obtained from the mouse genome database (accessed 2/3/15; Mouse Phenotype IDs 10425,10435,3105,10412,6113,285,10422,281,279,10429,2925) (S5 Table ). Cardiac development.Genes from the coexpression modules with correlation to one of the six developing cardiac tissues (atrioventricular canal, branchial arches, heart tube, atria, ventricle, and outflow tracts) were combined with RNAseq data from a key developmental structure in atrioventricular septal morphogenesis, the dorsal mesenchymal protrusion.These data combined yielded an early cardiac development gene set of 3,595 loci (S8 Table) [78]. Ocular malformations.To serve as a list of control genes unrelated to cardiac malformations, we developed a list of genes related to ocular malformations (n = 43) derived from the mouse genome database (accessed 7/15/15; Mouse Phenotype IDs 000516, 0002092) and the literature [79] and excluded genes appearing in the cardiac malformation gene list (S10 Table ). ",
"section_name": "Gene Lists",
"section_num": null
},
{
"section_content": "Live breeding pairs of the B6;129P2-Nr1d2 tm1Dgen /H mouse line (hereafter referred to as Nr1d2 tm1-Dgen ) were obtained from the European Mouse Mutant Archive (Munich, Germany).Animals were in housed and cared for in AAALAC accredited facilities under standard conditions with oversight and approval from the Stanford University APLAC committee (protocol APLAC-11334).Euthanasia was carried out under anesthesia with isofluorane following APLAC and AAALAC guidelines using carbon dioxide followed by cervical dislocation.Genotyping was performed by PCR of toe or tail clippings with gel electrophoresis using standard methodology with two primer pairs (CAAGTAACAAGCCTGGGACATAAAG and CTTCGTAGAGGGAGTAATATGACAC yield a 517 bp PCR product from the WT allele; CAAGTAACAAGCCTGGGACATAAAG and GACGAGTTCTTCTGAGGGGATCGATC yield a 757 bp product from the knockout allele).Two pairs of heterozygous founder animals were bred, yielding 17 pups (2 wild type, 7 Nr1d2 tm1-Dgen +/-, and 8 Nr1d2 tm1-Dgen -/-) which did not deviate obviously from expected Mendelian allelic ratios.Spontaneous death within hours after birth occurred in a single +/-and two -/-animals.The thoracic and abdominal cavities of spontaneously deceased homozygous knockout animals were visually inspected to examine the great vessels and visceral situs which were normal, and hearts dissected out and subject to sectioning and H & E staining by standard techniques.Sectioning of tissue samples was performed following either embedding of frozen sections followed by dehydration and fixation in 10% neutral buffered formalin or alternately fixation in 3% paraformaldehyde followed by alcohol dehydration and paraffin embedding.We performed additional matings of +/-and -/animals, and sacrificed mothers to obtain embryos at e16.5 and e17.5.Visualization was performed by brightfield microscopy on a Nikon 90i Eclipse upright with a DS Fi1 camera with a 20x objective.Both P0 Nr1d2 tm1-Dgen -/-animals displayed atrioventricular septal defects, a single -/-animal at e17.5 displayed an inlet ventricular septal defect, and single -/-animal at e16.5 displayed an AVSD.In total, 4 out of 15 -/-hearts assayed displayed a cardiac defect.Spontaneously deceased animals were analyzed, and additionally euthanasia of pregnant female mice was performed to obtain embryonic animals. ",
"section_name": "Mouse Experiments",
"section_num": null
},
{
"section_content": "The crystal structure of the DNA binding domain of NR1D1 (RCSB 1A6Y) was visualized with the PyMOL Molecular Graphics System, Version 1.7.4Schrödinger, LLC (New York, New York, USA).A construct containing wild-type murine Nr1d2 cDNA construct under control of a CMV promoter in the pCS6 expression vector was obtained from Transomic Technologies Inc. (Huntsville, Alabama, USA), and subject to a site-directed mutagenesis yielding a codon switch at p.R175W which corresponds to the de novo mutation observed in the subject with AVSD (S4a Fig).An Nr1d2 response element vector was constructed cloning 5 tandem repeats of an evolutionarily conserved NR1D2 binding site REV-DR2 [80,81] upstream of a minimal CMV promoter driving GFP expression using the pSF-MinCMV-daGFP vector (Sigma-Aldrich Inc, St. Louis, Missouri, USA) (S4b Fig) .Site-directed mutagenesis and cloning were performed by a commercial provider GENEWIZ Inc. (South Plainfield, New Jersey, USA), and sequence verified in our own laboratory.The three vectors were subject to routine endotoxin free preparation.Commercially available primary HUVEC cells obtained from Cell Applications (San Diego, California, USA) were seeded at 80% confluency in black transparent-flat bottom 96 well plates (Greiner, North Carolina, USA) and transfected the following day with 100ng of each vector (response-element, wild-type or p.R175W) using Lipofectamine 3000 (Life Technologies, Grand Island, New York, USA).Twenty-four hours after transfection, GFP fluorescence was measured on a Tecan Infinite M1000-multimode plate reader (Tecan Group Ltd, Mannendorf, Switzerland).We performed three transfection conditions including response-element+Nr1d2-WT, response-element+Nr1d2-P403W and response-element alone in 24 technical replicates.Autofluoresence of untransfected wells were averaged, and subtracted from the response-element alone and the two experimental conditions.The highest and lowest value from each condition were excluded from analysis yielding 22 technical replicates per experimental condition, and 4 technical replicates for the response element alone.Statistical analysis and graphing of the transfection experiments was performed in Prism 6 Graphpad Software (La Jolla, California, USA). ",
"section_name": "Cell Culture Experiments",
"section_num": null
},
{
"section_content": "S1 Text.Supplementary results.Additional phenotypic descriptions of individuals with AVSDs accompanied by mutations in syndromic genes or large CNVs.For AVSD patients displaying large CNVs (Table 2) or mutations in syndromic alleles, we returned to the medical record or referring provider to look for evidence of other syndromic features or developmental delay not ascertained at the time or enrollment.These phenotypic results are discussed herein.As noted in the Discussion, the two patients with compound heterozygous mutations in BBS2 were unavailable for follow up. ",
"section_name": "Supporting Information",
"section_num": null
}
] |
[
{
"section_content": "We thank Angela Rogers and Rick Dewey for thoughtful commentary on the manuscript.We thank SAP for funding a portion of the exome sequencing.We are grateful to all of the families at the participating Simons Simplex Collection (SSC) sites, as well as the principal investigators (A.Beaudet, R. Bernier, J. Constantino, E. Cook, E. Fombonne, D. Geschwind, R. Goin-Kochel, E. Hanson, D. Grice, A. Klin, D. Ledbetter, C. Lord, C. Martin, D. Martin, R. Maxim, J. Miles, O. Ousley, K. Pelphrey, B. Peterson, J. Piggot, C. Saulnier, M. State, W. Stone, J. Sutcliffe, C. Walsh, Z. Warren, E. Wijsman).We thank Janice Tracey for assistance with echocardiographic examinations.The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the NHLBI.We wish to posthumously recognize Ron Lauer MD of the University of Iowa for banking patient samples with congenital heart disease for future studies such as this one and to thank five anonymous reviewers for suggestions and critical evaluation of the manuscript.Our co-author Edward J. Lammer passed away unexpectedly during the review of this manuscript; we wish to acknowledge his lifelong contributions to the care and fundamental understanding of children with genetic disorders. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "Exome data can be found at the dbGAP repository maintained by NIH for researchers who meet the criteria for access to confidential data.Application procedures for dbGAP can be found at (https://dbgap.ncbi.nlm.nih.gov/aa/wga.cgi?page=login).Exome data from the Toronto single samples is freely available at the EGA repository for researchers who meet the criteria for access to confidential data.Application procedures for EGA cat be found at (https://www.ebi.ac.uk/ega/ about/access) Sage gene expression data from mouse organ development can be found at http:// ",
"section_name": "",
"section_num": ""
}
] |
10.1038/s41375-020-0747-7
|
Early treatment with FCR versus watch and wait in patients with stage Binet A high-risk chronic lymphocytic leukemia (CLL): a randomized phase 3 trial
|
<jats:title>Abstract</jats:title><jats:p>We report a randomized prospective phase 3 study (CLL7), designed to evaluate the efficacy of fludarabine, cyclophosphamide, and rituximab (FCR) in patients with an early-stage high-risk chronic lymphocytic leukemia (CLL). Eight hundred patients with untreated-stage Binet A disease were enrolled as intent-to-treat population and assessed for four prognostic markers: lymphocyte doubling time <12 months, serum thymidine kinase >10 U/L, unmutated IGHV genes, and unfavorable cytogenetics (del(11q)/del(17p)/trisomy 12). Two hundred and one patients with ≥2 risk features were classified as high-risk CLL and 1:1 randomized to receive either immediate therapy with 6xFCR (Hi-FCR, 100 patients), or to be observed according to standard of care (Hi-W&W, 101 patients). The overall response rate after early FCR was 92.7%. Common adverse events were hematological toxicities and infections (61.0%/41.5% of patients, respectively). After median observation time of 55.6 (0–99.2) months, event-free survival was significantly prolonged in Hi-FCR compared with Hi-W&W patients (median not reached vs. 18.5 months, <jats:italic>p</jats:italic> < 0.001). There was no significant overall survival benefit for high-risk patients receiving early FCR therapy (5-year OS 82.9% in Hi-FCR vs. 79.9% in Hi-W&W, <jats:italic>p</jats:italic> = 0.864). In conclusion, although FCR is efficient to induce remissions in the Binet A high-risk CLL, our data do not provide evidence that alters the current standard of care “watch and wait” for these patients.</jats:p>
|
[
{
"section_content": "Clinical observation without therapy-defined as \"watch and wait\" (W&W)-has been the gold standard for the management of early-stage chronic lymphocytic leukemia (CLL).This principle is based on the repeated failure of previous attempts to improve the clinical outcome of CLL patients by early therapeutic intervention [1][2][3][4].Moreover, a reasonable subset of patients with CLL experience an indolent disease course with neither compromising morbidity nor an elevated risk of premature death caused by the leukemia.Such patients have a life expectancy comparable with the normal population, and there is no justification to expose these cases to any potentially harmful antileukemic therapy [5][6][7]. However, there has still been a debate, whether cases with a more aggressive disease course could benefit from earlier treatment, in particular with the recent advent of targeted drugs.To date, reported trials that address the role of immediate therapy at an early disease stage have only tested single-agent chemotherapies (i.e., chlorambucil and fludarabine), but no modern treatment options, such as combined chemoimmunotherapy or novel small-molecule inhibitors. The study presented here (named \"CLL7\" trial) was aimed at testing whether chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab (FCR) would improve the outcome of patients with unfavorable prognosis when administered at an early stage.FCR has been the first regimen to prolong survival of advanced-stage CLL, and represents a standard of care option for first-line treatment of physically fit CLL patients [8][9][10][11][12].We present data of a German-French collaborative phase 3 trial that compared early FCR therapy versus \"watch-and-wait\" in Binet A patients with the categorized high-risk CLL disease.We implemented an advanced four-parameter risk stratification system, including genetic disease features to prospectively segregate cases with the Binet A high-risk CLL from those with the low-risk disease, and to direct their therapeutic management in a randomized fashion. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "A prospective randomized phase 3 trial (CLL7) was collaboratively conducted by the German CLL study group (GCLLSG) and the French Cooperative Group on CLL (FCGCLL).Patients with early-stage CLL were registered at 69 sites in Germany, Austria, and Switzerland, and 25 sites in France, in case the following main inclusion criteria had been fulfilled (Supplementary Table 1): diagnosis of CLL according to NCI-working group criteria [13], established not earlier than 12 months prior to registration, Binet stage A disease, no prior treatment, age ≥ 18 years, and Eastern Cooperative Oncology Group performance status 0-2.Patients with clinically evident autoimmune cytopenias, active second malignancies or infections, longterm use of steroids, or other severe medical illnesses or organ dysfunctions were not eligible.All patients provided written informed consent before registration.The trial was conducted according to the Declaration of Helsinki, and approved by ethical review boards responsible for each of the participating centers.It was registered at the US National Institute of Health (NCT00275054) and the EU clinical trial database (EudraCT 2005-003018-14). ",
"section_name": "Trial design and participants",
"section_num": null
},
{
"section_content": "After registration, the following risk parameters were assessed in central laboratories of the GCLLSG and FCGCLL according to standard protocols: serum thymidine kinase (TK) levels, the mutation status of the immunoglobulin heavy-chain variable region genes (IGHV), and recurrent chromosomal abnormalities by fluorescence in situ hybridization.The lymphocyte doubling time (LDT) was calculated by regression curve analysis from a minimum of three lymphocyte counts obtained in at least 4-week intervals within 6 months before registration.Risk factor results were collected at the German and French biometry centers, respectively (Institute for Medical Statistics and Epidemiology (IMSE), Technical University of Munich, Germany; Department de Biostatistiques et Informatique Medicale, Hôpital Saint Louis, Paris, France), where for each patient the final risk evaluation and stratification/randomization procedures were performed.Patients with at least two of four adverse prognostic markers present (TK > 10 U/ L, LDT < 12 months, IGHV unmutated, or deletion (del) in chromosome 11q or 17p, or trisomy 12) were categorized as high-risk (Hi) patients, while patients with <2 of these markers present were categorized as low risk (Lo).High-risk patients were one-to-one randomized to either receive FCR chemoimmunotherapy (Hi-FCR) or being observed (Hi-W&W) using a previously generated randomization list (IMSE).The randomization was balanced by the use of randomly permuted blocks with a block size of four, and was stratified according to country and number of adverse prognostic markers.Low-risk patients were only assigned to clinical observation (Lo-W&W). ",
"section_name": "Risk stratification and randomization",
"section_num": null
},
{
"section_content": "Patients randomized to the Hi-FCR arm were assigned to receive a maximum of six cycles of intravenous FCR, given in 28-day intervals.Fludarabine (25 mg/m 2 ) and cyclophosphamide (250 mg/m 2 ) were administered on day 1-3 of each cycle.Rituximab was given at 375 mg/m 2 on day 0 of cycle 1, and at 500 mg/m 2 on day 1 of cycles 2-6.According to the protocol, the prophylactic use of growth factors was left to the discretion of the local investigator.In case of grades 3-4 neutropenia with signs of a concurrent infection, the administration of G-CSF was mandatory per protocol.Anti-infective prophylaxis with trimethoprim/ sulfmethoxazole was recommended from day 1 until the end of 2 months after the last dose of the last cycle.Additional details on parental drug administration, concomitant medication, and dose reduction rules are described in Supplementary Methods. Baseline disease assessment included physical examination, ECOG performance status, assessment of B symptoms and comorbidity, imaging of disease manifestations via ultrasound or computed tomography (CT), laboratory assessments from peripheral blood (PB) including parameters routinely assessed prior to the administration of cytoreductive therapies, serum beta-2-microglobulin, and lymphocyte immunophenotyping.Patients underwent baseline and follow-up disease assessments at month 4 (interim staging after three cycles of therapy, Hi-FCR only), month 8 (final staging after therapy), and 12, in 6-month intervals between months 12 and 60, and once per year thereafter.Response assessment after FCR therapy included routine clinical and laboratory assessments, radiographic imaging of CLL manifestations (used method at the discretion of the local investigator), and flow cytometry for minimal residual disease (MRD) assessment.The latter was performed using fourcolor flow cytometry for the German and six-color flow cytometry for the French cohort.For further details refer to Supplementary Methods [14,15].A uniform threshold was applied to define MRD negativity as less than one detected CLL cell per 10,000 leukocytes analyzed per flow cytometry.After treatment completion, a bone marrow (BM) aspirate/ biopsy was recommended per protocol in case the patient achieved a complete remission (CR). ",
"section_name": "Patient treatment and procedures",
"section_num": null
},
{
"section_content": "The primary objective of the study was to compare the efficacy of early versus deferred FCR in Binet stage A patients at high risk for disease progression.The secondary objective was to prospectively validate the prognostic value of the above-mentioned four-parameter risk stratification system for Binet A patients.The primary endpoint was event-free survival (EFS), considering progression, treatment, or death as events.Among the secondary endpoints were overall response rate (ORR), overall survival (OS), progression-free survival (PFS), adverse events related to treatment, molecular response, response duration, and time to (re)treatment (TTT).The toxicity of FCR treatment was determined according to the Common Terminology Criteria (CTC) for Adverse Events version 3.0.The response status after FCR therapy and disease status during follow-up was evaluated according to the NCI-working group criteria [13]. ",
"section_name": "Outcomes",
"section_num": null
},
{
"section_content": "Details on the sample size computation for this study, data responsibilities, and data sharing are described in Supplementary Methods.The primary analysis was a two-sided logrank test that was stratified by country and number of risk factors in a second step to confirm the results.Time-to-event endpoints were estimated according to the Kaplan-Meier method.Survival curves were compared using nonstratified log-rank tests.Hazard ratios (HR), including 95% confidence intervals (CI), were calculated by Cox regression analysis under the assumption of proportional hazards.Exploratory post hoc subgroup analyses were done considering MRD status, IGHV mutational status, and cytogenetic categories.All tests were two sided, and a p value < 0.05 was considered significant.Adjustments for multiple testing were not done.Safety analyses were restricted to patients from the intention-to-treat population who received at least one dose of one component of the study treatment (safety population).ORR was calculated based on both the intention-to-treat and on the safety population.Statistical analyses were performed using SPSS v23 (SPSS, Chicago/IL, USA). ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Between 2005 and 2010, a total of 824 patients were registered for the CLL7 study, 423 in 69 GCLLSG centers in Germany (51.3%), Austria, and Switzerland, and 401 (48.7%) in 25 centers of the FCGCLL in France.After exclusion of patients, who did not fulfill the study requirements, and completion of risk assessment, 800 patients (ITT population), aged 27-81 years, were stratified into 201 high-risk (25.1%) and 599 low-risk (Lo-W&W) patients (74.9%) (Fig. 1).The median time from registration to risk stratification was 3 months (0-29.1 months).One hundred and one high-risk patients were randomized to the observation arm (Hi-W&W), while the remaining 100 patients were allocated to receive early FCR (Hi-FCR).Both high-risk arms were well balanced with respect to country, age, comorbidity, ECOG status, white blood count (WBC), IGHV mutation status, trisomy 12, and del(17p) (Table 1).There was an imbalance in the prevalence of elevated TK, short LDT, male sex (each more common in Hi-FCR), and del(11q) (more common in Hi-W&W) between the two high-risk cohorts.B symptoms and lymphadenopathy as signs of a more aggressive disease course were more common in high-risk than in low-risk patients. ",
"section_name": "Study population",
"section_num": null
},
{
"section_content": "Eighty-two percent of 100 Hi-FCR patients received at least one dose of FCR, and were included in the safety analysis (safety population).Eighteen percent patients withdrew their consent for early therapy after randomization and before FCR had been initiated.The median number of administered treatment cycles was 6 (range, 1-6) and 67 patients (81.7%) completed six cycles of study therapy.The documented reasons for discontinued FCR (<6 cycles, 15 patients, 18.3%) were hematotoxicity (6 patients, predominantly neutropenia), fever/infections (2 patients; 1 CMV reactivation, 1 infection of unknown origin), consent withdrawal or allergic exanthema (2 patients each), 1 hospitalization due to rupture of an aortic aneurysm, 1 thrombosis with consecutive pulmonary embolism, and 1 autoimmune hemolytic anemia (AIHA). In 20 patients (24.4%) at least one study drug was dose reduced >20% in one or more cycles.Most frequently, the doses of fludarabine and cyclophosphamide were reduced (15 cases) due to hematologic toxicity (11 cases with at least one of the following events: 9 leukopenia/neutropenia, 2 thrombocytopenia, 1 cytopenia not further specified, and 1 anemia).Other reasons for dose-reduced FC were febrile infections (two cases), inpatient treatment due to a ruptured aortic aneurysm (one case), a collapse during infusion (one case), or unknown (two cases).Dose-reduced rituximab was given in five patients (twice unintentionally by missing the rituximab dose increase at cycle 2, in two cases for unknown reasons, and in one case due to an event of bradycardia). Overall, 203 grade 3-5 adverse events in 61 patients (74.4% of safety population) were reported.In addition, there were 18 events documented in five patients, without sufficient information (including missing CTC grade) available.One hundred and twenty-five of those 203 events (86.2%) were categorized as at least possibly related to the study treatment by the local investigator.The three most common categories were hematotoxicity (50 patients, 61.0% of safety population; most frequently leukopenia/neutropenia), infections (18 patients, 22.0%), and metabolic/laboratory events (5 patients, 6.1%; most frequently elevated liver enzymes) (Table 2).Recurrent types of infections were respiratory tract infections (seven patients, 8.5% of the safety population), fever/infections of unknown origin (three patients, 3.7%), herpes zoster reactivations (three patients, 3.7%), and catheter-related infections (two patients, 2.4%).Use of growth factor support with G-CSF was documented in 25 out of 82 FCR treated patients (30.5%). There were two of total four fatal adverse events during follow-up, documented with a potential relationship to administered FCR: one patient succumbed 9.3 months from stratification to a suspected viral encephalitis (clinical/radiologic diagnosis), which was judged as possibly related to the study therapy.Another patient died 9.8 months from stratification due to a persisting AIHA that had occurred under FCR therapy.Two additional deaths were documented at month 41.8 and 53.2 from stratification with no relationship to study therapy.Causes of death were reported as a pulmonary fibrosis and progressive renal failure in the context of a Richter's transformation, respectively. In the overall study, we did not detect an elevated risk of disease transformation, second(ary) malignancies, or AIHA ",
"section_name": "Early FCR treatment and safety",
"section_num": null
},
{
"section_content": "The overall response to early FCR based on the ITT population according to NCI-working group criteria [13] was 76.0% (76 out of 100 patients allocated to the Hi-FCR arm).Out of 82 patients, who had received at least one dose a Cumulative illness rating scale [32]. b ECOG = performance status scale according to the Eastern Cooperative Group [33].c According to Döhner et al. [24].d Three patients were allocated to the incorrect risk stratum according to their risk profile presented here.Two of those cases (one Hi-W&W and one Lo-W&W) were caused by entry/capture errors for assigned risk factors in the database; and, these patients were stratified in the correct risk subset.Only one Lo-W&W patient was truly misstratified as a low-risk case, despite the fact that two risk factors had been found present by central diagnostics. of FCR (safety population), 76 (92.7%) achieved a remission (Table 3).Twenty-seven patients (32.9%) obtained a BM confirmed CR, 34 patients (41.5%) an at least clinical CR without BM evaluation, and 15 patients (18.3%) obtained a partial remission (PR).In three patients, response assessment was missing, but they had received only one or two cycles of the study therapy, respectively.Three patients (3.8%) had stable disease after therapy.In two of those three cases treatment had been stopped prematurely after one or two cycles due to grade 3 neutropenia and grade 3 febrile neutropenia, respectively.The highest CR rates were achieved in patients who underwent at least three cycles of therapy, were IGHV mutated, or carried a del(11q).Fifty-three and 28 Hi-FCR patients were available for MRD assessment by four-color flow cytometry from PB and BM, respectively.Forty of 53 patients (75.5%) were MRD negative (≤10 -4 ) in PB at the time of final response assessment, 13 patients (24.5) were MRD positive (>10 -4 ).In BM, 67.9% (19 out of 28 patients) achieved an MRDnegative remission. The primary endpoint, EFS, was significantly prolonged in high-risk patients treated with an early FCR (Hi-FCR) versus deferred treatment according to the current standard of care (Hi-W&W).After a median follow-up of 55.6 months (range 0-99.2 months), only 36 patients (36.0%) in the Hi-FCR arm had progressed, received new CLL therapy, or died, compared with 83 patients (82.2%) in the Hi-W&W arm (median EFS not reached in Hi-FCR vs. 18.5 months in Hi-W&W, HR 0.22, 95% CI 0.15-0.33,p < 0.001 for stratified and nonstratified log-rank test) (Fig. 2a).High-risk patients with a MRD-negative response to early FCR in PB significantly benefited from the quality of remission with regard to EFS compared with patients with an MRD-positive response (landmark analysis, median EFS not reached versus 41.2 months, log-rank p < 0.001, HR 10.68, 95%CI 3.51-32.55,Fig. 3a; for MRD from BM refer to Supplementary Fig. 1).Twelve Hi-FCR and 11 Hi-W&W patients had died.In both studies, arms major causes of death were infections and progressive disease including Richter's transformation (Supplementary Table 3).There was no significant OS benefit for high-risk patients receiving early versus deferred FCR (5-year OS 82.9% in Hi-FCR vs. 79.9% in Hi-W&W, HR 0.93, 95% CI 0.41-0.22,p = 0.864, Figs.2b and3b, Supplementary Fig. 2). After 5 years from the last dose of study medication, 80.4% of Hi-FCR patients (safety population) had not received any further treatment for CLL (median TTT not reached, Fig. 4b), compared with 21.8% of patients in the Hi-W&W arm (Fig. 4a). Our risk stratification system successfully segregated patients with differential prognosis.High-risk patients with or without early FCR therapy (Hi-FCR/Hi-W&W) demonstrated a significantly shorter EFS, PFS, and OS than patients categorized with the low-risk disease (Fig. 2a,b, Supplementary Fig. 3).Briefly, patients assigned to Hi-W&W had a 8.0 times higher risk of progression, treatment or death (HR 8.02, 95%CI 6.04-10.65,p < 0.001), while Hi-FCR patients had a 1.8 times higher risk (HR 1.82, 95% CI 1.26-2.63,p = 0.002) of progression, treatment or death, compared with the Lo-W&W cohort.Corresponding 5-year EFS rates were 12.6%, 55.2%, and 77.1% in Hi-W&W, Hi-FCR, and Lo-W&W patients, respectively.In total, 87.1% of Lo-W&W patients were without treatment at 5 years from stratification (median time to first-line treatment 27.6 months in Hi-W&W vs. not reached in Lo-W&W, HR 11.62, 95% CI 8.23-16.39,p < 0.001).Patients with the Fig. 3 Event-free survival (EFS) and overall survival (OS) according to MRD status in peripheral blood.a EFS from MRD landmark (final response assessment/MRD evaluation).b OS from MRD landmark (final response assessment/MRD evaluation).MRD minimal residual disease.For this calculation, the MRD status at the final restaging was considered.MRD negative < 10 -4 ; positive ≥ 10 -4 detected CLL cells per leukocytes, according to MRD-flow cytometry. For MRD-results from bone marrow please refer to Supplementary Figs. 1 and2. low-risk disease demonstrated an excellent survival at 97.2% 5 years from stratification (HR Hi-FCR/W&W vs. Lo-W&W 5.82, 95% CI 2.98-11.36,p < 0.001) (Fig. 2b). ",
"section_name": "Efficacy of early FCR and survival",
"section_num": null
},
{
"section_content": "We present data of a phase 3 trial (CLL7), which successfully implemented molecular genetic disease characteristics into a risk-tailored treatment allocation strategy for patients with stage Binet A CLL. Twenty-five percent of our ITT study population exhibited a \"high risk\" disease type according to our four-factor risk assessment, and these patients clearly segregated from the low-risk group with regard to all time-to-event parameters investigated, i.e., illustrated by EFS, PFS (Supplementary Fig. 3), and OS.All four risk parameters used for our study design were chosen due to their confirmed value as prognostic factors for PFS/OS in multivariate analyses performed in the first 147 patients registered in the preceding CLL1 trial (phase 3 comparison of fludarabine vs. W&W in Binet A CLL) in 2004 [2].In particular, we found serum levels of the TK (cutoff 10 U/L) rather than beta-2-microglobulin (3.5 mg/L) as a preferred independent prognostic factor for time-toevent outcome in our test set analysis, and therefore implemented serum TK in our study design [16].The parameter LDT reflects the disease dynamics, and is recommended by current guidelines to determine the right time a patient requires therapy [17].Particularly at an early disease stage, an LDT < 12 months has been identified as an independent indicator of an unfavorable prognosis [18][19][20].Although easily assessable in clinical practice, the parameter is not commonly documented in large trial datasets, and therefore not considered in the latest CLL scoring systems, such as the CLL-IPI [21][22][23]. The scientific background to include trisomy 12 as a risk factor in our stratification approach was formed by the hierarchical model, developed by Döhner et al. before this study was designed [24].Recent long-term follow-up data in FCR studies, however, demonstrated that patients with trisomy 12 have a particularly favorable PFS/OS after FCR, when treated at an advanced disease stage [9,25,26].Thus, in retrospect it might have been specifically difficult to achieve further improvement for this patient population with our early treatment strategy (Supplementary Figs. 4 and 14). A comparative analysis of our risk stratification and the CLL-IPI as a current standard risk assessment in CLL is included as Supplementary Table 4.It indicates that the CLL7 stratification between low-risk and high-risk subsets correponds to a segregation between CLL-IPI low risk versus CLL-IPI intermediate/high/very high risk in the majority of cases. The data presented here demonstrate that an early application of FCR was able to postpone events of disease progression and the need of therapy in stage Binet A highrisk CLL, but despite this effect, there was no OS benefit in the long run.FCR was highly effective in reducing the tumor load in treated patients, as demonstrated by a high OR and CR rate.Moreover, while the significance of the MRD data set is limited by a relatively low number of assessments, the frequency of achieved MRD negativity (PB: 75.5%, BM: 67.9%) compares favorably to the respective data from the FCR arm of the CLL8 trial (63% and 44%, respectively) [27].Patients who achieved a MRD-negative status (at a threshold of 10 -4 in PB) appeared to enjoy a better prognosis (median EFS not reached) than previously reported for MRD-negative patients with active disease treated within the CLL8 trial (median PFS 64 months).These findings underline not only the important ability but also potentially higher likelihood of disease-eradicating activity by treatment regimens applied at an early disease stage. The shortcomings of this study might be the primary endpoint EFS from stratification may be criticized for not considering the difference in the disease load in early treated versus observed patients, and hence, for implementing an upfront advantage or disadvantage, respectively, in the risk of progression.It should be considered that this study was initiated at a time when the clinical experience with FCR, used at an advanced disease stage, was still limited to make projections on outcome for a study design like ours.We preferred to choose a primary endpoint, which commences at trial outset for all patients, most independent from other dynamic variables, and which allows a study design realistic to be accrued.Not all patients in the Hi-W&W arm did receive FCR as a deferred frontline therapy.Per protocol, the use of FCR was recommended, in case Hi-W&W patients were in need of therapy.According to collected data on the choice of first-line therapy in the Hi-W&W arm (available in 70 patients, Supplementary Table 5), the use of anti-CD20 treatment was a common choice made for first-line therapy in the Hi-W&W arm, but also use of less efficacious treatments (i.e., R-CHOP, obinutuzumab + chlorambucil, various monotherapies) were given.In addition, the application of new oral kinase/small-molecule inhibitors at later disease stages in the overall high-risk population might have influenced the survival data as they are. It could be argued that an elevated risk to die from treatment-related early or late toxicity might have mitigated any survival benefits in the Hi-FCR arm.In comparison to other studies investigating frontline FCR at an advanced disease stage, our study did not clearly detect a significantly higher or unexpected toxicity of FCR, when administered at an early stage.For example, the documented rate of CTC grade 3/4 hematotoxicity after deferred FCR was 56% in the CLL8 trial (phase 3 registration study for FCR versus fludarabine plus cyclophosphamide (FC)) [9].In the FCR arm of the CLL10 study (phase 3 study on FCR versus BR) [28], grade 3/4 neutropenia occurred in 84% of patients, the overall rate of patients with grade 3 hematological events was 21% and 69% for grade 4, respectively.We observed grade 3/4 infections in 22% of treated patients in the Hi-FCR arm compared with 25% of patients treated with FCR in CLL8, and 35% (grade 3) and 3% (grade 4) of patients treated with FCR in CLL10.The use of growth factors was not generally recommended in all of these protocols and not equally documented for a head-to-head comparison.Further, the causes of death documented in both high-risk arms of our study-predominantly progressive CLL disease and infections-did reveal an increased mortality by late adverse treatment effects.Although a direct comparison of toxicity rates between different trials has to be interpreted with caution, these data allow the conclusion, that the tolerability of FCR in our study was comparable to what has been experienced with its use in advanced-stage CLL.A mandatory use of growth factors like G-CSF might have been adequate to limit the rate of neutropenia and the associated risk of infections. To rule out a particular hazard of an early FCR in a distinct molecular subset of patients we also compared timeto-event outcome according to the IGHV mutation status, and in cytogenetic subsets [24] (Supplementary Figs.[4][5][6][7][8][9][10][11][12][13][14][15].No particular benefit or disadvantage of early versus late therapy could be detected in these subgroups with respect to EFS and OS.Although not statistically significant due to low patient numbers, there was a particular adverse disease course in three of four early treated patients with del(17p), who died within 12.2 months from stratification (Supplementary Fig. 13).The causes of death were persisting AIHA, a cerebral stroke, and hemophagocytosis/infectious complications after allogeneic stem cell transplant, respectively. Molecular genetic studies in advanced CLL have revealed a high level of clonal heterogeneity and ongoing genetic evolution of CLL cells throughout the disease course and in particular under applied treatment pressure [29,30].Clinically, clonal evolution might have become evident in the Hi-FCR arm of our trial with lower remission rates or response durations after second-line therapies.These data were not the focus of this trial or analysis.However, those considerations warrant careful monitoring of molecular alterations evolving under ongoing treatment pressure, and their consequences on sequential treatment outcome in future studies in early-stage CLL. In conclusion, FCR therapy is feasible in Binet A stage CLL and extends EFS and PFS in patients with high-risk disease.As a caveat of early FCR we observed possibly treatment-related deaths in 2.4% of treated patients.In accordance with previous treatment studies in an early-stage CLL, our trial does not provide any evidence that the significant improvement of EFS in this patient population translates into a survival benefit.Therefore, \"watch & wait\" after diagnosis, until \"active disease\" criteria [31] are met, remains the standard of care, irrespective of unfavorable prognostic features.Ongoing and future studies may elucidate, whether the immediate use of such targeted and potentially disease-eradicating therapies (i.e., venetoclax combinations), will be able to overcome adverse disease courses (particularly for patients with del(17p)), and to displace the current standard of care \"watch & wait\" [11]. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "Acknowledgements We are indebted to the participating centers and investigators, as well as all patients who contributed to the completion of this study.For the GCLLSG we acknowledge Henrik Gerwin, Gabriele Kranz, Silke Schott, and Irene Stodden for their work as project and/or data managers for the study, Sabine Frohs for her activity as safety manager.The database of the study was generated with the support of WiSP (Wissenschaftlicher Service Pharma GmbH, Langenfeld, Germany).Monitoring of participating centers was performed with support of WiSP and the Competence Net Malignant Lymphoma (KML), Germany.This was an investigator initiated trial which was sponsored by the University of Cologne and the FCGCLL.The trial was primarily funded by the German Cancer Aid (106142/ 107080) and the French National Research Agency (ANR).Additional funding support, as well as the study drug rituximab was provided by Roche Pharma AG, Grenzach, Germany/Boulogne-Billancourt Cedex, France.Subprojects with respect to detailed analyses of infections and molecular profiling of patients included in the study were funded by the German Ministry for Education and Research (01KI0771/ 01KI101) and the German Research Foundation (KFO286).SS was supported by European Commission/BMBF (\"FIRE CLL\", 01KT160) and Deutsche Forschungsgemeinschaft (SFB 1074, project B1, B2).All funders did not have any involvement in the study design, collection, analysis and interpretation of data, or writing of the report.Open access funding provided by Projekt DEAL. ",
"section_name": "Compliance with ethical standards",
"section_num": null
},
{
"section_content": "Acknowledgements We are indebted to the participating centers and investigators, as well as all patients who contributed to the completion of this study.For the GCLLSG we acknowledge Henrik Gerwin, Gabriele Kranz, Silke Schott, and Irene Stodden for their work as project and/or data managers for the study, Sabine Frohs for her activity as safety manager.The database of the study was generated with the support of WiSP (Wissenschaftlicher Service Pharma GmbH, Langenfeld, Germany).Monitoring of participating centers was performed with support of WiSP and the Competence Net Malignant Lymphoma (KML), Germany.This was an investigator initiated trial which was sponsored by the University of Cologne and the FCGCLL.The trial was primarily funded by the German Cancer Aid (106142/ 107080) and the French National Research Agency (ANR).Additional funding support, as well as the study drug rituximab was provided by Roche Pharma AG, Grenzach, Germany/Boulogne-Billancourt Cedex, France.Subprojects with respect to detailed analyses of infections and molecular profiling of patients included in the study were funded by the German Ministry for Education and Research (01KI0771/ 01KI101) and the German Research Foundation (KFO286).SS was supported by European Commission/BMBF (\"FIRE CLL\", 01KT160) and Deutsche Forschungsgemeinschaft (SFB 1074, project B1, B2).All funders did not have any involvement in the study design, collection, analysis and interpretation of data, or writing of the report.Open access funding provided by Projekt DEAL. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "",
"section_name": "Compliance with ethical standards",
"section_num": null
},
{
"section_content": "Conflict of interest CDH received research funding and travel support from Roche.PL and KF received travel support from Roche.JB received consulting honoraria and travel support from Roche.MH and CMW report advisory board membership for Roche, and obtained research funding, consulting honoraria, and speaker's honoraria from Roche.SB reports consulting fees, honoraria, and research funding from Roche.SS declares advisory board membership, consulting honoraria, speaker's honoraria, research grants and travel support from Roche.All other authors declare no competing financial interests. 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": "",
"section_num": ""
}
] |
10.1038/s41467-019-10363-1
|
Structures of BCL-2 in complex with venetoclax reveal the molecular basis of resistance mutations
|
<jats:title>Abstract</jats:title><jats:p>Venetoclax is a first-in-class cancer therapy that interacts with the cellular apoptotic machinery promoting apoptosis. Treatment of patients suffering chronic lymphocytic leukaemia with this BCL-2 antagonist has revealed emergence of a drug-selected BCL-2 mutation (G101V) in some patients failing therapy. To understand the molecular basis of this acquired resistance we describe the crystal structures of venetoclax bound to both BCL-2 and the G101V mutant. The pose of venetoclax in its binding site on BCL-2 reveals small but unexpected differences as compared to published structures of complexes with venetoclax analogues. The G101V mutant complex structure and mutant binding assays reveal that resistance is acquired by a knock-on effect of V101 on an adjacent residue, E152, with venetoclax binding restored by a E152A mutation. This provides a framework for considering analogues of venetoclax that might be effective in combating this mutation.</jats:p>
|
[
{
"section_content": "T he connection between aberrant cell death and cancer 1 sparked a detailed understanding of the mitochondrial pathway to apoptosis 2 and led to the first BCL-2 antagonist approved for cancer therapy, venetoclax 3,4 .BCL-2 is the founding member of the pro-survival class of proteins that includes BCL-X L , BCL-W, A1/BFL-1 and MCL-1.They exert their pro-survival function by binding and restraining related members of a family of pro-apoptotic proteins-the sensors of cellular stress (the BH3only proteins) and the effectors of apoptosis (BAX and BAK).This restraint is exerted through interactions between the helical BCL-2 Homology 3 (BH3) motifs of pro-apoptotic molecules and a cognate groove on the surface of pro-survival proteins 5 .Four or more hydrophobic amino acids on successive helical turns within the BH3 motif engage complementary pockets (P1 through P4) in this surface groove of pro-survival proteins.Blocking this interaction by targeting the binding groove with organic ligands has long held promise for cancer therapy 6,7 , and venetoclax is the first realisation of that promise. The first bona fide BH3 mimetics, ABT-737 and ABT-263 targeted multiple pro-survival family members BCL-2, BCL-X L and BCL-W, engaging with the P2 and P4 pockets in their BH3binding groove 6,8 .Treatment with these compounds results in thrombocytopenia, an on-target toxicity due to BCL-X L inhibition, which limits their use as effective chemotherapeutics 9 .This led to a revised strategy of selectively targeting BCL-2 that resulted in ABT-199 (venetoclax) 10 .Venetoclax spares platelets but retains the ability to promote apoptosis in malignant cells dependent on BCL-2.It is among the first approved small molecule cancer therapeutics that directly blocks a proteinprotein interaction 11 .Other BH3 mimetics are in development, including another BCL-2 selective compound, S55746 12 . Mutation of drug-binding sites is a common mechanism by which malignant cells evade therapies, as typified by resistance to the ABL1 tyrosine kinase inhibitors 13 .Resistance to some tyrosine kinase inhibitors can be conferred through mutation of threonine 315 to isoleucine (T315I).Rational drug design strategies have been successful in developing approved therapeutics that are effective in cases with the T315I mutation 14 .Venetoclax was approved in 2016 and as a consequence relatively few patients have been treated to date and there is a limited understanding of potential resistance mechanisms.To predict potential resistance mutants a mouse model was used to induce venetoclax tolerance in cancer cells 15 .This study identified that mutation of phenylalanine 104 (human numbering) to either leucine (F104L) or cysteine (F104C), located within BCL-2's BH3-binding groove, rendered the lymphoma cell line resistant to venetoclax.Subsequent work confirmed that these mutations can also confer resistance in models of human leukaemia and lymphoma 16 , but have not yet been observed in patients.Recently a novel BCL-2 mutation was described exclusively in patients undergoing treatment with venetoclax 17 .This mutant, G101V, was found in chronic lymphocytic leukaemia (CLL) patients from the clinical trials who had initially responded to treatment but developed CLL-type clinical progression after 19-42 months 17 .Tellingly, the presence of the mutation in patient samples was predictive of clinical progression.The BCL-2 G101V mutation reduces the affinity for the drug to BCL-2 by some 180-fold.On the other hand BCL-2 G101V maintains affinity for the BH3 motif of pro-apoptotic proteins, such as BAX and BIM, and thus can still function to suppress apoptosis.By selectively reducing affinity to venetoclax the BCL-2 G101V mutation provides resistance to the therapy.To understand how the G101V and other resistance mutations can selectively decrease the affinity for venetoclax whilst retaining anti-apoptotic activity we searched for a structural rationalisation. Currently 18 BCL-2 structures (4 NMR and 14 crystal structures) are deposited in the protein data bank (PDB) 6,10,12,[18][19][20][21][22][23][24] . The first disclosure of venetoclax (ABT-199) in 2013 included several structures of compounds bound to BCL-2, including the structure of ABT-263 (PDB:4LVT) and an analogue of ABT-199 (PDB:4MAN) 10 , but no structure of venetoclax itself with BCL-2 has yet been published. Here we describe crystal structures of venetoclax bound to wild type BCL-2 and the BCL-2 mutants G101V and F104L.We also characterise the binding profiles of the drug to various BCL-2 mutants by surface plasmon resonance.Through these analyses we reveal the molecular mechanisms by which these mutants compromise drug-binding and, in the case of G101V, enable disease progression.These structures pave the way for rational optimisation of the venetoclax scaffold to counter this BCL-2 mutation. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Crystal structure of BCL-2 bound to venetoclax.Crystal structures of BCL-2 with ABT-263 and various analogues of venetoclax have been deposited in the PDB and described in the literature (Fig. 1a, b) 10 .One of those analogues is 4- amino]phenyl)sulfonyl)benzamide, hereafter referred to as compound 1.We obtained crystals of BCL-2 in complex with venetoclax that diffracted to high resolution (1.62 Å) in the space group P2 1 2 1 2 1 with one molecule in the asymmetric unit (Fig. 1a,b, Table 1).The electron density for the drug was well defined (Supplementary Fig. 1a,b) and the binding pose was in general agreement with the published structures of ABT-263 and compound 1, with the 4-chlorophenyl (CP) bound in the BCL-2 P2 pocket, the piperazine bridging the P2 and P4 pockets over residue F104 and the azaindole substitution bound in the BCL-2 P4 pocket.It was possible to model two distinct conformations with the 4-4-dimethylcyclohex-1-ene (4DM) ring flipping at the 4 and 5 positions above the BCL-2 P2 pocket and the benzamide (BA) acyl oxygen adopting two conformations above the BCL-2 P4 pocket (Fig. 1b).An interesting difference was the positioning of the venetoclax 4DM moiety, which was further from the α4 helix and more central over the P2 pocket than the equivalent rings from ABT-263 and 4MAN compounds (Fig. 1c,d, Supplementary Fig. 2, Supplementary Table 1).This small deviation in the positioning of the 4DM was unexpected as the equivalent six membered ring systems from ABT-263 and compound 1 are similar, differing only by the positioning of the gem-dimethyl group in ABT-263 (5 position) and the lack of a methyl and a phenyl ring in compound 1 (Fig. 1a). Structures of BCL-2 mutants bound to venetoclax.To understand how these BCL-2 mutations compromise drug binding we solved crystal structures of both complexes (Table 1 and Fig. 2).The G101V mutation resides on the BCL-2 α2 helix packing against the α5 helix and is within the BCL-2 BH3 motif.The glycine is a conserved, defining feature of the motif.Adjacent residues A100 and D103 define boundaries of the P4 pocket so the mutation was expected to alter drug binding by changes to this region (Fig. 2a).The BCL-2 G101V:venetoclax complex crystallised in P2 1 spacegroup with two molecules in the asymmetric unit diffracting to 2.2 Å, with well-defined electron density for both copies of the drug (Supplementary Fig. 1).The overall structure of BCL-2 G101V was similar to WT with no significant deviations in α2-α5 core helices or α6-α8 (Fig. 2b,c).There was a minor change in orientation of the α1 helix resulting in a ~1 Å deviation at either end of the helix, but this was far from the drug binding site.The binding pose of venetoclax is conserved between WT and the G101V mutant (Fig. 2b).The P2 pocket volume was maintained at 478 Å 3 (480 Å 3 for WT) as was the volume of the P4 pocket at 379 Å 3 (380 Å 3 for WT).Interestingly, the G101V mutation did not alter the positioning of the α2 helix relative to α5 or impact the residues defining the P4 pocket.Instead the additional bulk of the valine sidechain was accommodated by a deviation of the sidechains of Y18 on α1 and E152 on α5 (Fig. 2c).In the BCL-2 G101V:venetoclax structure E152 had a 60º rotamer change relative to WT (mm-40 for WT to tp10 for G101V), placing the sidechain Cγ in van der Waal's contact with the chlorine atom of the venetoclax chlorophenyl moiety.This conformational change in E152 in the BCL-2 G101V structure causes a small repositioning of the venetoclax 4DM and chlorophenyl moieties in the P2 pocket, moving on average 0.25 Å closer to L137 in the α4 helix (Fig. 2b, Supplementary Fig. 2, Supplementary Table 1), i.e. more similar to the BCL-2 WT complexes with ABT-263 and compound 1.Additionally, we obtained a structure of BCL-2 G101A bound to venetoclax (Table 1), representing a milder introduction of bulk at the G101 position than the valine substitution.The BCL-2 G101A:venetoclax and BCL-2 WT:venetoclax crystals were isomorphous and there were no significant deviations in venetoclax positioning or E152 (Supplementary Fig. 2,3, Supplementary Table 1).Despite the closer proximity of the azaindole moiety to the mutation site, its orientation in the P4 pocket was conserved between BCL-2 WT and G101V structures.Therefore, the G101V mutation appears to modulate venetoclax affinity more through its interactions with the P2 pocket than the P4 pocket. The crystals of venetoclax complexed with BCL-2 F104L and BCL-2 WT are isomorphous (Table 1).Well-defined electron density for the drug in the mutant complex structure (Supplementary Fig. 1) suggests two conformations for the 4DM and acyl group of the BA moiety as in WT (Fig. 2d).The side chain of F104 separates the P2 and P4 pockets of BCL-2.The P4 pocket volume was maintained between WT and F104L structures (P4 pocket volume 380 Å 3 for both WT and F104L).In contrast the volume of the P2 pocket increased with the F104L mutation as the leucine sidechain occupies a smaller volume than phenylalanine (Fig. 2d-f).In this structure two conformers for F112 on α3 are evident, one like WT and a second occupying some of the volume vacated by the F104L mutation.(Fig. 2c, e andf).The new F112 conformation extends into the P2 pocket, packing against L104.This inserted F112 conformation compensates for the loss of P2 pocket volume in the BCL-2 F104L mutant and is comparable to the BCL-2 WT P2 pocket volume-P2 pocket volumes of 480 Å 3 (BCL-2 WT), 475 Å 3 (F104L inserted conformation) and 596 Å 3 (F104L conserved conformation).The occupancy of F112 refined to 0.48 for the conserved conformation and 0.52 for inserted conformation, indicating that the compensation in P2 pocket volume only occurs 50% of the time and the vacated space is unfavoured.The consequence of the F104L mutation is to alter the packing environment of the chlorophenyl moiety of the drug. Binding of BH3 peptides and venetoclax to BCL-2 mutants.SPR experiments were performed using a BIMBH3 or BAXBH3 immobilised sensor surface with BCL-2 mutants as the analyte and determining venetoclax affinity by competition experiments 17,25 , (Fig. 3, Table 2 and Supplementary Fig. 4,5).We have previously reported BIMBH3 and BAXBH3 affinities for WT, G101V and F104L 17 , and these were comparable to F104C, with less than 10-fold change relative to WT (Table 2).In contrast, the affinities for venetoclax differed by 25 to ~1500-fold with K I values 0.018, 3.2, 0.46 and 25 nM for WT, G101V, F104L and F104C, respectively (Table 2).This indicates that the BCL-2 mutants maintain tight binding to BH3 domains, allowing their overexpression to prevent apoptosis, whilst selectively reducing the affinity for the drug and thus providing resistance to therapy. The role of E152 in venetoclax affinity.E152 moved into the base of the P2 pocket in the BCL-2 G101V:venetoclax structure (Fig. 2b,c).To test the role of E152 in reducing affinity we generated a BCL-2 G101V/E152A double mutant.Alanine does not have a Cγ or Cδ to impact the base of the P2 pocket and would allow the chlorophenyl to insert unimpeded into the P2 pocket in the G101V mutant.We repeated SPR experiments with the BCL-2 G101V/E152A double mutant and a BCL-2 E152A single mutant (Table 2 and Fig. 3c,d).The E152A single mutant had comparable binding to WT and when combined with G101V as a double mutation restored high affinity venetoclax binding, with WT binding at 18 pM, BCL-2 E152A at 27 pM and BCL-2 G101V E152A at 2 pM (Table 2 and Fig. 3c,d).The BCL-2 G101V/E152A affinity was 10-fold higher than WT, however competition SPR experiments become less accurate as the ligand K I becomes significantly tighter or weaker than the K D for the competing BimBH3 peptide; as such it is unclear whether this increase in affinity is significant.Furthermore, the E152 conformation in a BCL-2 G101A:venetoclax structure matched the WT conformation, not the G101V.The G101A mutant bound to venetoclax with a K I of 110 pM comparable to WT but distinct from G101V (Table 2 and Supplementary Fig. 4).This indicates that E152A mutation rescues high affinity for venetoclax when combined with G101V and confirms the importance of the E152 rotamer change observed in the G101V mutant.Note also that the affinity of BCL-2 G101V/E152A and BCL-2 E152A for BIMBH3 and BAXBH3 was largely unaltered compared to WT (Table 2 and Supplementary Fig. 3). BCL-2 G101V binding to S55746.S55746 is another BCL-2 selective antagonist that has progressed to the clinic.The recently disclosed crystal structure of BCL-2 WT bound to S55746 revealed binding to the P1, P2 and P3 pockets 12 , in contrast to venetoclax that binds principally to the P2 and P4 pockets (Fig. 4).We tested the binding of S55746 to both BCL-2 WT and G101V by competition SPR (Table 2, Fig. 3 and Supplementary Fig. 6).S55746 bound to BCL-2 WT with a K I of 0.36 nM and G101V with a 100-fold lower K I of 36 nM, in both cases > 10-fold weaker than venetoclax, K I of 0.018 nM and 3.2 nM for WT and G101V, respectively.This was confirmed in cellular assays using the B-lineage cell line KMS-12-PE (Fig. 4a).BCL-2 WT and G101V were overexpressed in the KMS-12-PE cells and S55746 LC50 concentrations were determined as 0.32 ± 0.15 μM for WT increasing eight-fold to 2.7 ± 0.43 μM with the G101V mutation. To further investigate this we solved the structure of BCL-2 G101V bound to S55746 (Table 1).We obtained diffraction to 2.0 Å in a P 2 1 spacegroup with two molecules in the asymmetric unit.The BCL-2 G101V:S55746 structure was in general agreement with the published structure of the BCL-2 WT: S55746 complex PDB ID 6GL8 (Fig. 4b,c) 12 .The P2 pocket is key for interactions with BH3 domains 26,27 , which typically display a leucine residue engaging this pocket.S55746 inserts a 4hydroxyphenyl moiety into the P2 pocket similar to the chlorophenyl of venetoclax.In that case the chlorophenyl inserts deeply into the P2 pocket causing F112 to change rotamer (venetoclax rotamer t80, S55746 rotamer m-85) and exposing E152 at the base of the P2 pocket (Fig. 4d).In the S55746 structure F112 seals the P2 pocket shielding the S55746 hydroxyphenyl from E152 (Fig. 4d).In the BCL-2 WT:S55746 structure E152 is in the tp10 rotamer configuration similar to the BCL-2 G101V: ABT-199 structure.However, in the BCL-2 G101V: S55746 structure E152 is in an unconventional rotamer, which shows most similarity to tp10, with the Cγ deviating by 40º from the conventional tp10 rotamer (Fig. 4d).We tested binding of the BCL-2 G101V/E152A double mutant to S55746 giving a K I of 5.3 nM.This was over 10-fold higher than WT (K I of 0.32 nM), but 10-fold lower than G101V (K I of 36 nM), indicating partial but not complete rescue of S55746 affinity with the double mutant (Fig. 3g).Thus, one cause of the reduction in affinity of S55746 for BCL-2 G101V can be traced to the knock-on effect of the V101 side chain against E152. ",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Currently venetoclax is approved for treatment of patients with previously treated chronic lymphocytic leukemia 4 .Venetoclax selectively inhibits BCL-2, thereby promoting apoptosis in cells refractory to conventional apoptotic cues.No crystal structure for BCL-2 binding to venetoclax has been described, although analogues have been published 10 .We have now crystallised and determined the structure of venetoclax bound to BCL-2 at high resolution.The structure reveals subtle differences between the binding of the drug relative to the published analogues (Fig. 1), which would not have been predicted based on the previous structures.Notably, the orientation of the moiety inserted into the P2 pocket is subtly different in venetoclax compared to ABT-263 and compound 1 structures.The P2 pocket is an important determinant in selectivity of BH3 peptides and BH3 mimetics 26,28 , and here it emerges as the critical feature conferring resistance to a drug-selected BCL-2 mutant, G101V.The BCL-2 G101V mutation was exclusively identified in CLL patients with disease progression on venetoclax clinical trials, but only after many months of continuous treatment.Early detection of BCL-2 G101V by highly sensitive ddPCR assays predicted subsequent clinical disease progression.The mutation reduces the affinity of the drug for BCL-2 by ~180-fold 17 .In contrast, it only moderately reduces the affinity for BH3 peptides, allowing the mutant to still function normally as a pro-survival protein.Interestingly, G101 is part of the BCL-2 BH3 sequence.The BH3 motifs of BH3-only proteins or of BAX and BAK are ligands for a binding groove on pro-survival BCL-2 proteins, but the role of the BH3 motif in pro-survival proteins is unclear.Notably, the motif consensus sequence of ϕ1-x-x-x-ϕ2-x-x-ϕ3-G-D-x-ϕ4, where ϕ1-4 are hydrophobic amino acids, includes the largely conserved G101 of BCL-2.There are examples of BCL-2 family proteins that have either alanine or serine instead of glycine at this position, but not valine.In the multi-BH domain BCL-2 proteins the BH3 motif is in the α2 helix, with the small glycine packing against the BH1 domain in the α5 helix.The G101V mutation is adjacent to but not directly part of the P4 pocket that engages a leucine residue (ϕ4) when the BAX BH3 motif binds (Fig. 5).We could not obtain crystals for BCL-2 G101V bound to BAXBH3 peptide so cannot comment on how the mutation affects BAXBH3 interactions in this region.The azaindole ring of venetoclax, one of the key features of its selectivity for BCL-2, occupies this pocket.However, there were no differences in the P4 pocket with venetoclax bound to BCL-2 or BCL-2 G101V.Instead the additional bulk from the valine sidechain is accommodated by movements in the positions of Y18 (α1) and E152 (α5), with a distinct rotamer change for the glutamate.The E152 rotamer change in the BCL-2 G101V:venetoclax complex places the Cγ sidechain atom in contact with the chlorophenyl introducing a subtle change in the orientation of that moiety in the P2 pocket.To illustrate the connection between these small structural movements and the ~180-fold reduction in affinity for the G101V mutant, we introduced the E152A mutation on the G101V mutant and restored near-wildtype affinity for venetoclax.Additionally, introduction of an alanine at the G101 position did not provide sufficient bulk to displace E152 in the crystal structure and affinity for venetoclax was maintained for the G101A mutant.Thus the functional consequences of the G101V mutant are felt in the P2 pocket via the 'knock-on' effect of E152 repositioning forced by the additional bulk of the valine sidechain. The BCL-2 F104L and F104C mutations were observed as venetoclax-resistance mutations in a mouse tumour model 15 and both induce drug tolerance in human cell lines 16 .Interestingly, in the initial study, human cell lines did not acquire resistance through mutation to BCL-2, instead truncation of BAX occurred preventing translocation to the mitochondria 15 .The BCL-2 F104L mutation has been observed in non-Hodgkin lymphoma 29 suggesting the mutation is viable in lymphomas, however these patients were not treated with drug and to date neither the F104L or F104C mutations have been reported in patients receiving venetoclax therapy.Here we have shown through competition SPR experiments that neither the BCL-2 F104L nor F104C mutants suffer significant reduction in binding to the BH3 peptides of pro-apoptotic BAX or BIM.In contrast, venetoclax binding decreased by ~10-500-fold (for F104L and F104C, respectively) relative to WT BCL-2.The structure of BCL-2-F104L:venetoclax presented here reveals that the P2 pocket increases in volume with the F104L mutation relative to WT (480 Å 3 and 596 Å 3 for WT and F104L, respectively).This increase in pocket volume decreases the surface complementarity between the drug and its target, likely contributing to the decrease in affinity.Binding to the F104C mutant is weaker still, and this may have thwarted attempts to obtain a crystal structure of the BCL-2 F104C:venetoclax complex despite extensive efforts.This further decrease in affinity is likely due to an even larger P2 pocket volume, as cysteine occupies a smaller volume than leucine, though other structural features may also come into play. Pro-survival proteins prevent apoptosis by binding and sequestering the pro-apoptotic proteins.Venetoclax competes with pro-apoptotic BH3 motifs for BCL-2 binding, releasing proapoptotic proteins and allowing apoptosis in cells primed for death 6,8 .It is therefore required that drug-resistant mutants of BCL-2 retain the ability to bind pro-apoptotic BH3 motifs to maintain the tumour's viability.The G101V, F104L and F104C BCL-2 mutants all have this property.The critical difference between venetoclax and a BH3 helical peptide is the greater penetration of the drug into the P2 pocket, with the G101V mutant this compromises drug-binding but not BH3-binding.This is also a feature of ABT-737, one of the earliest precursors of venetoclax 26 .Analogues of venetoclax that may retain binding to BCL-2 G101V include those that lack the chlorine of the chlorophenyl moiety or have it replaced with a smaller atom.S55746 binds BCL-2 via the P1, P2 and P3 pockets and our SPR data show it binds to BCL-2 WT with 10-fold lower affinity than venetoclax.S55746 inserts a 4-hydroxyphenyl moiety into the P2 pocket but does not insert as deep into the pocket as the corresponding chlorophenyl from venetoclax.This shallow insertion allows the BCL-2 F112 on the α3 helix to insert into the BCL-2 groove extending the P3 pocket.We show here that S55746 also loses potency (~100-fold) against the BCL-2 G101V mutant and we sought a structural explanation for this.The orientation of S55746 in the BCL-2 G101V:S55746 complex structure is conserved relative to the WT structure.In both BCL-2 WT and BCL-2 G101V structures compared with S55746 E152 is shielded from the base of the P2 pocket by the insertion of F112 into the groove.Consistent with this the combination of the G101V mutation with the E152A mutation did not fully restore WT binding to S55746, in contrast to venetoclax, suggesting additional structural features underlying affinity in this case.Those features may include changes in the structure and dynamics of BCL-2 G101V prior to engaging drug, or the distribution of conformers in the ensemble of BCL-2 G101V:drug structures in solution compared to WT that are not readily detectable crystallographically 30 . Venetoclax is the first FDA-approved drug that directly targets the mitochondrial apoptotic pathway.We have solved the crystal structure of the drug bound to its target, BCL-2, revealing a pose subtly different to that observed with structural analogues.We also describe the molecular basis for resistance to venetoclax observed in patients on treatment displaying the BCL-2 mutant G101V.These structures provide a basis to further optimise the venetoclax scaffold, for binding both to BCL-2 WT and the G101V mutant. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Compounds, peptides and primers.All peptides were custom synthesized by Mimotopes Pty Ltd (Australia).S55746 was purchased from ProbeChem (Cat#PC-63502).Venetoclax was purchased from ActiveBiochem (#A-1231).All other chemicals, specified, were obtained from Sigma-Aldrich (Australia).Primer sequences are detailed in Supplementary Table 3. Protein expression and purification.BCL-2 WT construct, expression and purification was reported previously 31 and the G101V and F104L mutants were previously reported 17 .Briefly, mutants were introduced by PCR using primers with the desired mutation.Bacterial E. coli BL21 cells were transformed with appropriate plasmids and proteins expressed as N-terminal GST fusions by IPTG induction in SuperBroth.Recombinant GST-BCL-2 fusions were purified from cellular proteins by glutathione-agarase resin (Genscript; CAT#L00206) and eluted with 10 mM reduced glutathione.GST-BCL-2 fusions were cleaved overnight at 4 °C with Prescission Protease to remove the GST-fusion and purified to homogeneity by size exclusion chromatography using a Superdex 75 10/300 (GE healthcare) equilibrated in 20 mM Tris pH 8 and 150 mM NaCl. Crystallisation.BCL-2 proteins were incubated with 3 molar excess of the desired compound in DMSO.DMSO was removed by buffer exchange in an Amicon® Ultra-4 10 kDa cut of spin concentrator (Millipore; CAT#UFC801096).Initial crystals were obtained with BCL-2 WT and the desired compound by the hanging drop vapour diffusion method at 291 K with a precipitant solution consisting 5% PEG4K, 40% PEG400, 0.1 M MES pH 6.0.Initial BCL-2 WT: venetoclax analogue crystals were used as crystallisation seeds to streak seed, using a cat's whisker, into a new crystallisation drop containing BCL-2 WT and venetoclax to obtain BCL-2 WT:venetoclax crystals.All subsequent crystallisation experiments with mutants and venetoclax were obtained by streak seeding from BCL-2 WT:venetoclax crystals.Crystallisation conditions were optimised by diluting precipitant concentrations by a factor in the range 0.5-1 with water and varying the pH by 0.2 intervals from the initial condition.BCL-2 G101V:S55746 crystals were obtained by the hanging drop vapour diffusion method with a precipitant solution consisting 1 M NaCl, 0.1 M Succinic acid-NaOH pH 5.0, 10% PEG200.Prior to diffraction experiments crystals were cryoprotected by supplementing the mother liquor with PEG400, glycerol or ethylene glycol, then cooled in liquid nitrogen. Structure determination.All datasets were collected at 100 K at the Australian Synchrotron MX2 beamline using an ADSC quantum 315R CCD detector (BCL-2 WT:venetoclax and BCL-2 F104L:venetoclax only) or an Eiger X 16M direct detector (all other datasets) 32 .Diffraction experiments were processed in XDS 33 and scaled in either XDS or Aimless 34 .The phase problem was solved by molecular replacement using Phaser 35 and the BCL-2 WT:navitoclax structure chain A with all waters and ligands removed (PDBid 4LVT) as a search model 10 .The model was refined by iterative reciprocal and real space refinement using PHENIX refine 36 and Coot 37 , respectively with at least one round of cartesian simulated annealing refinement in PHENIX prior modelling ligands to avoid phase bias.Ligand initial models and restraints for venetoclax and S55746 were generated using the Grade web server (version 1.2.9,Global Phasing Ltd.).For atoms with multiple conformations initial occupancies were set to 0.5 modelling each conformer into appropriate density prior to multiple rounds of refinement in phenix using the refine occupancy function in addition to standard refinement procedures.X-ray data to geometry weights or atomic displacement factors were determined automatically using the optimize X-ray/stereochemistry weight and optimise X-ray/ ADP weight respectively.Model validation was performed in Coot and Mol-Probity 38 .Data statistics were calculated in PHENIX using the generate Table 1 for journal function.Stereo images with electron density are displayed in Supplementary Fig. 7. The BCL-2 G101V:venetoclax data processed and solved in the orthorhombic spacegroup P2 1 2 1 2 1 with a single protein chain in the assymetric unit.However, during refinement R fact and R free increased in successive refinement cycles, giving final values that were unreasonable when compared to similar resolution structures from the protein data bank.This did not occur when the structure was modelled in the monoclinic spacegroup P2 1 with two protein chains in the assymetric unit.As a consequence, the monoclinic model and data were used. The BCL-2 G101V:S55746 structure processed in the monoclinic spacegroup P2 1 with two protein chains in the assymetric unit.The data were anisotropic with data extending to 2.7 Å in the a* and 2.0 Å in the b* and c* directions, and were ellipsoidally truncated and scaled by the diffraction anisotropy server 39 without applying B-factor sharpening.After applying ellipsoidal truncation the data completeness in high resolution shells dropped, with completeness dropping below 90% from 2.5-2.0Å.Furthermore, the electron density for S55746 in one of the protein chains was more poorly resolved.This copy of S55746 was modelled into the electron density according to the S55746 orientation from the BCL-2 WT: S55746 structure (PDB id 6GL8) 12 .To avoid phase bias no coordinates from the original WT:S55746 structure were used in refinement and orientations were matched by protein alignments and visual inspection only.The analysis presented here relates to the protein chain without this problem. Structural analyses.All crystal structure representations were made using Mac-PyMOL version 1.8.0.3 (Schrödinger LLC).BCL-2 pocket volumes were calculated in a multistep step process.Initially PDB models were stripped of all non-protein atoms and pockets were filled with water molecules using the hollow program (version 1.2) 40 with a 12 Å sphere radius from residue BCL-2 104 and 0.2 grid spacing.BCL-2 pockets were then defined as any water molecule within 5 Å of the venetoclax chlorophenyl (atoms CL and C10) for the P2 pocket or 2 Å from atoms in the venetoclax azaindole (atoms C37-43, N5-6).Water atom selections were visually inspected to remove atoms that may be included in selection criteria but not connected to the desired pocket.The volume of each pocket selection was then determined using the 3vee web server volume assessor function (http://3vee.molmovdb.org/volumeCalc.php) 41 ",
"section_name": "Methods",
"section_num": null
}
] |
[
{
"section_content": "Research in the authors' laboratory is supported by NHMRC project grants (1059331 and 1079706), program grants (11131233 DCSH, PMC; 1113577 AWR) and fellowships (1156024 DCSH, 1079560 AWR, 1116934 PMC, 1079700 PEC), the Leukemia and Lymphoma Society (SCOR 7015-18 and Fellowship 5467-18 to RT), the Victorian Cancer Agency (Fellowship to IJM), project support from The Cancer Council of Victoria (Project 1124178 to IJM), the Victorian State Government Operational Infrastructure Support, and the Australian Government NHMRC IRIISS.This research was undertaken in part using the MX2 beamline at the Australian Synchrotron, part of ANSTO, and made use of the Australian Cancer Research Foundation (ACRF) detector.The authors would like to thank Yan-Hong Tan for technical assistance, Dr W. Douglas Fairlie and Dr Erina Lee for providing BCL-2 plasmid DNA and the Collaborative Crystallisation Centre (C3, CSIRO) for crystallisation screening.We would also like to thank Wayne Fairbrother for discussions related to the manuscript.Finally the authors would like to thank the following cats and their owners for donating their naturally shedded whiskers for crystallisation seeding experiments: Tilly, Boris, Patsy and Snoop Catty Cat. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "The refined coordinates and data for all structures were deposited in the PDB: BCL-2: venetoclax PDB id 6O0K, BCL-2 G101V:venetoclax PDB id 6O0L, BCL-2 G101A: venetoclax PDB id 6O0P, BCL-2 F104L:venetoclax PDB id 6O0M and BCL-2 G101V: S55746 PDB id 6O0O.The source data underlying Fig. 3a-h, Fig. 4a and Table 2 are provided as a Source Data File.A reporting summary for this Article is available as a ",
"section_name": "Data availability",
"section_num": null
},
{
"section_content": "(91-164).In the BCL-2:venetoclax structures there is a crystal contact between R18 and the acyl oxygen from the venetoclax benzamide (a hydrogen bond) in one of the alternate conformations (conformer A).The conformation of the venetoclax conformer A benzamide is equivalent to the conformation of ABT-263 bound to BCL-2 (PDB id 4LVT) which has no crystal contacts to the bound ligand.The published BCL-2 WT:compound 1 structure (PDB id 4MAN) has a crystal contact between the α5-6 loop and the benzine ring from the benzamide moiety of compound 1 from symmetry related molecules that may influence the conformation of compound 1. Distances venetoclax cyclohex-1-ene ring atoms (C1-6), ABT-263 atoms (C30, C39, C29, C26, C25 and C28 ordered according to equivalent positions on venetoclax) or compound 1 (C1, C9, C29, C23, C21 and C28) ordered according to equivalent positions on venetoclax) and BCL-2 L137 Cα in Supplementary Table 1 were determined using the distance command in MacPyMOL version 1.8.0.3.For fair comparison the BCL-2 WT:venetoclax cyclohex-1-ene ring A conformer was used as a reference as it matched the conformers from the ABT-263 and G101V:venetoclax structures.The BCL-2 WT: ABT-263 (PDB id 4LVT), BCL-2 WT:compound 1 (PDB id 4MAN) and BCL-2 G101V:venetoclax structures had two copies of protein and drug in the asymmetric unit.Distances for both copies were calculated and mean values presented in the Supplementary Table 1.RMSD values were calculated for each structure relative to the BCL-2 WT:venetoclax cyclohex-1-ene ring A conformer. Surface plasmon resonance.SPR experiments were performed as previously described 17 .Briefly, experiments were performed in HBS-EP buffer consisting 10 mM hepes pH 7.4, 150 mM sodium chloride, 3.4 mM EDTA, 0.005% tween 20 and optionally 1 mM TCEP for BAXBH3 experiments, at 25 °C.Experiments were perfomed on either a BIAcore 4000 or BIAcore S200 using a SA sensor chip (GE healthcare) immobilized with biotinylated BIMBH3 (DMRPEIWIA-QELRRIGDEFNAYYARR) or BAXBH3 (ADASTKKLSECLKRIGDELDSN-MELQRMIAA) peptide, using a BIMBH3-4A peptide (DMRPEIWAAQEARRAGDEANAYYARR) as a non-binding reference.BIMBH3 and BAXBH3 Peptide affinities were determined by direct binding with BCL-2 (0-63 nM) as the analyte.The lowest concentrations used in direct binding experiments were 1 nM for BIAcore 4000 and 0.1 nM for BIAcore S200 instruments, concentrations below these were below the sensitivity thresholds for the instruments.Venetoclax affinity was determined by competition against immobilized BIMBH3 peptide, using BCL-2 (0-250 nM) pre-mixed with venetoclax (0, 20, 40, 60 nM) as the analyte.Direct binding experiments were fitted to a 1:1 binding site kinetic model, fitting on and off rates, in BIAevaluation software.For competition experiments steady-state responses were determined in BIAevaluation software according to the software defaults in affinity evaluation mode averaging responses for 5 sec.Response data were fitted to a steady-state competition model (Equation 1) in Prism 7.0d for mac (GraphPad Software, La Jolla California USA): peptide apparent steady-state binding constant, K I = fitted equilibrium binding constant for venetoclax (or S55746).BIMBH3 K D and R max for the steady-state competition model were calculated in Prism 7.0d for mac using the data from 0 nM venetoclax from the same experiment, using a one-site specific binding model.All affinity measurements were performed in at least two independent experiments using independent protein preparations. Plasmids, retrovirus production and infection.Wild-type BCL-2 construct was reported previously 31 .Point mutation at BCL-2 Gly101 to Val was introduced using primers (with the desired mutation) using PCR.cDNA encoding either wildtype FLAG-BCL-2 or -BCL-2 mutants were inserted into the MSCV-IREShygromycin retroviral construct as previously described 42 . Cell lines.KMS-12-PE (sourced from DSMZ in 2013; Cat#ACC606) were cultured with HTRPMI with 10% fetal bovine serum.Early passages (P5-P7) after purchase were cryopreserved and thawed for the experiments.Cells beyond passage 15 were not used.Monthly tests for mycoplasma were consistently negative (MycoAlert mycoplasma detection kit; Lonza, GA, USA).The expression level of WT or G101V BCL2 in KMS-12-PE cells were determined by intracellular FACS using a BCL2 antibody (BCL2-100, WEHI, 1:200) 43 on an LSR-Fortessa flow cytometer. Cell viability assays.To test the sensitivity of the engineered KMS-12-PE inhibitor to S55746, cells were seeded in a 96-well plate at 5000 cells/well and treated with serially diluted concentrations (0-10 μM, 5-point 1:8 dilution) of drug.Cell viability 24 h after treatment was then determined using the CellTiter-Glo assay (Promega, Cat#G9241).Experiments were performed on antibiotic-resistant pools of cells transfected with vectors expressing wild-type, mutant BCL-2 or the empty vector control.Experiments were performed on three separate occasions; the data is shown as the means ± SD of those three independent experiments. ",
"section_name": "",
"section_num": ""
}
] |
10.3390/jcm10040867
|
Aberrant Expression of TLR2, TLR7, TLR9, Splicing Variants of TLR4 and MYD88 in Chronic Lymphocytic Leukemia Patients
|
<jats:p>Functional toll-like receptors (TLRs) could modulate anti-tumor effects by activating inflammatory cytokines and the cytotoxic T-cells response. However, excessive TLR expression could promote tumor progression, since TLR-induced inflammation might stimulate cancer cells expansion into the microenvironment. Myd88 is involved in activation NF-κB through TLRs downstream signaling, hence in the current study we provided, for the first time, a complex characterization of expression of TLR2, TLR4, TLR7, TLR9, and MYD88 as well as their splicing forms in two distinct compartments of the microenvironment of chronic lymphocytic leukemia (CLL): peripheral blood and bone marrow. We found correlations between MYD88 and TLRs expressions in both compartments, indicating their relevant cooperation in CLL. The MYD88 expression was higher in CLL patients compared to healthy volunteers (HVs) (0.1780 vs. 0.128, p < 0.0001). The TLRs expression was aberrant in CLL compared to HVs. Analysis of survival curves revealed a shorter time to first treatment in the group of patients with low level of TLR4(3) expression compared to high level of TLR4(3) expression in bone marrow (13 months vs. 48 months, p = 0.0207). We suggest that TLRs expression is differentially regulated in CLL but is similarly shared between two distinct compartments of the microenvironment.</jats:p>
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is a disease with the accumulation of aberrant B cells in peripheral blood as well as proliferation and accumulation of CLL cells in the bone marrow and peripheral lymphoid organs.CLL patients are characterized by different prognoses, as well as profound molecular and immune defects [1][2][3].Immune deregulations result in high susceptibility to infections as well as a failure to improve effective antitumor immune responses [4][5][6].The nonspecific immune response could be continuously stimulated in CLL patients, although it has been suggested that the stimulation of toll-like receptors (TLRs) expressed on CLL cells could increase the immunogenicity of tumor cells and thus potentially contribute to the induction of tumor-specific immune response [7,8]. TLRs represent a family of transmembrane receptors that recognize a broad spectrum of pathogen-associated molecular patterns (PAMPs), such as highly conserved structures of viral (TLR7 or TLR9), bacterial (TLR2 or TLR4), and endogenous molecules released by injured tissues [8].TLRs regulate innate immunity and determine the polarization and function of adaptive immunity mediated by B and T cells.TLRs expression is highly up-regulated through B-cell receptor (BCR) triggering of naive B-cells, indicating synergism between TLR and BCR leading to B-cell proliferation as well as differentiation [9].All TLRs downstream signaling pathways except TLR3 are conducted by adaptor molecule Myd88 (myeloid differentiation primary response protein 88).It has been proven that TLRs types including 5, 7, 8, 9, 11 initiate the Myd88-dependent pathway directly.However, Myd88-dependent signaling pathway through TLRs types 1, 2, 4, 6 involves TIR-domaincontaining adaptor protein (TIRAP).TLR3 and TLR4 initiate an alternative pathway which is MyD88-independent by recruiting TIR-domain-containing adaptor protein, inducing IFN-β (TRIF) [10].Activation of MyD88 plays a crucial role in inflammatory cytokine secretion.The MyD88-dependent signaling pathway leads to the early phase of nuclear factor-κB (NF-κB) activation, whereas the MyD88-independent signaling pathway initiates the late phase of NF-κB activation.TLR-dependent signals could be also involved in the regulation of B lymphocytes function by inducing TLR tolerance or auto-reactivity promotion [11].TLR7 and TLR9 engaged together with BCR also participate in the response to auto-antigens [12].Recent studies indicated that both auto-and exogenous antigens might be engaged in the initiation and progression of CLL.The majority of data emphasized the role of adaptive immune receptors, including BCR, in the pathogenesis and progression of the disease [13][14][15]. The consequence of the occurrence of the MYD88 mutation includes chronic activation of TLRs, thus the constitutive activation of the NF-κB promotes cell proliferation and survival [16].Recent studies confirmed the prognostic value of MYD88 mutation in CLL [17].Since the MYD88 mutation is harboring in 1-10% CLL patients, the current study aimed to characterize the association between expression and mutational status of MYD88 and TLRs expression in the bone marrow and peripheral blood in CLL. Splicing variants of TLRs might have different abilities to induce signal transduction since alternative splicing produces various transcripts and, as a result, various proteins from a single gene [18].The current study aimed to present an expression pattern of TLR2, TLR7, TLR9, and splicing variants expression of TLR4 (TLR4(1), TLR4(3), TLR4(4)) on the mRNA level as well as perform a comparison in two different microenvironmental compartments, peripheral blood and bone marrow, referring to recognized prognostic markers as well as clinical outcome. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "The material was obtained from 94 untreated CLL patients diagnosed at the Department of Hematooncology and Bone Marrow Transplantation, the Medical University of Lublin (60 males, 34 females, mean age 65).In the current study, we included patients with CLL diagnosed by aberrant immunophenotype, including CD5+ CD19+.The median value of CD19+ CD5+ expression analyzed on peripheral blood mononuclear cells (PBMC) was always >90%. Thirty-three patients were classified in stage A, thirty-four in stages B, and eleven in stages C, according to the Binet classification.Detailed characteristics of CLL patients are shown in Table 1.This material was also obtained from 27 healthy volunteers.This study was approved by the Local Ethics Committee (KE-0254/7/2019), and the patients were informed about the use of their blood for scientific purposes. ",
"section_name": "Study Subjects",
"section_num": "2.1."
},
{
"section_content": "Mononuclear cells from peripheral blood (PBMC) and bone marrow (BMMC) were isolated by Ficoll (Biochrom AG, Berlin, Germany) density gradient.Next, cells were washed twice in phosphate-buffered saline (Biochrom AG, Berlin, Germany) and counted.The viability of obtained PBMC and BMMC was always >95%, as determined by trypan blue exclusion (Sigma-Aldrich, Schnelldorf, Germany).The viable cells were quantified in a Neubauer chamber (Zeiss, Jena, Germany) and stored for RNA preparation at -192 • C in liquid nitrogen. ",
"section_name": "Isolation of Mononuclear Cells",
"section_num": "2.2."
},
{
"section_content": "For the isolation of mRNA from PBMC, the QIAamp RNA Blood Mini Kit (Qiagen, Venlo, The Netherlands) was used according to the manufacturer's instructions.One µg of mRNA was reverse transcribed into 20 µL of cDNA using a QuantiTect Reverse Transcrip-tion Kit (Qiagen, Venlo, The Netherlands).For each RT-PCR, 1 µL of the cDNA preparation was used.2.4.Quantitative Polymerase Chain Reaction (q-PCR) For the quantitative measurement of the mRNA expression of TLR2, TLR4, TLR7 and TLR9, real-time q-PCR was performed using the Light Cycler SYBR Green I technology according to the manufacturer's protocol (Roche Diagnostics, Rotkreuz, Switzerland).The sequences of the primers for q-PCR are shown in Table 2 (Roche Diagnostics, Rotkreuz, Switzerland). ",
"section_name": "RNA Isolation and Reverse Transcription",
"section_num": "2.3."
},
{
"section_content": "Sequences of Primers The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as a housekeeping gene.An initial denaturation step at 95 • C for 10 min was followed by 40 cycles of 15 s at 95 • C and 1 min at 60 • C. The qPCR reactions were carried out using 7300 Real Time PCR System (Applied Biosystems, Foster City, CA, USA). The TLRs mRNA expression was calculated as an inverse ratio of the difference in cycle threshold (∆Ct), where ∆Ct is the Ct value of the target gene minus Ct value of GAPDH. For the quantitative measurements of the mRNA expression of MYD88, qPCR was performed using TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assay primer/probe mixes (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions.GAPDH was used as a reference gene.Thermocycling program was set for 40 cycles of 15 s at 95 • C and 1 min at 60 • C on the 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).The MYD88 mRNA expression was calculated as an inverse ratio of the difference in cycle threshold (∆Ct), where ∆Ct is the Ct value of the target gene minus Ct value of GAPDH. ",
"section_name": "Type of TLR (Splicing Variants)",
"section_num": null
},
{
"section_content": "The MYD88 L265P mutation was analyzed in 61 CLL patients in blood samples using the three primers: the mutant-specific reverse primer-5 -CCT TGT ACT TGA TGG GGA ACG-3 , the wild-type-specific reverse primer-5 -GCC TTG TAC TTG ATG GGG AAC A-3 , and the common forward primer-5 -AAT GTG TGC CAG GGG TAC TTA G-3 .Two reverse primers were designed to differentiate the mutant and wild-type allele of MYD88 L265P before the PCR reaction DNA isolation was performed using QIAamp DNA Blood Mini Kit (Qiagen, Venlo, The Netherlands) according to the manufacturer's instructions.PCR was performed in a total reaction volume of 25 µL with 10 pmol of each primer and 100 ng DNA using QIAGEN Multiplex PCR Kit (Qiagen, Hilden, Germany).Thermal cycling conditions were: 2 min of preheating at 94 • C followed by 40 cycles of 94 • C for 30 s, 57 • C for 30 s, and 68 • C for 30 s, with a final extension at 68 • C for 5 min.The PCR products (159 bp) were separated on 2% agarose gel electrophoresis and visualized under UV light. ",
"section_name": "Detection of the MYD88 L265P Mutation by the Allele Specific-Polymerase Chain Reaction (AS-PCR)",
"section_num": "2.5."
},
{
"section_content": "Statistical analysis was performed using GraphPad Prism 5.All results are presented as median values with rage.The U Mann-Whitney test and Kruskal-Wallis test was used to evaluate the difference between subgroups of patients.The correlations of variables were computed with Spearman's rank correlation coefficient.Survival curves were calculated for time to first treatment (TTFT) of CLL patients according to the Kaplan-Meier method using a log-rank test.TTFT was calculated in months since the date of initial diagnosis until the date of initial treatment.For the subgroup of patients who had never received any treatment, we characterized TTFT as the date of the last follow up. ",
"section_name": "Statistical Analysis",
"section_num": "2.6."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "The expression of TLR2, TLR7, TLR9 and splicing variants of TLR4 was confirmed in PBMC in CLL patients as well as in healthy volunteers (HVs).The expression of TLR2 in peripheral blood was found to be lower in CLL patients compared to HVs with a median 0.2185 vs. 0.2632 (p = 0.039) (Figure 1A).Similarly, the expression of splicing variants of TLR(4), including TLR4(1) and TLR4(4), was significantly lower in CLL patients than in HVs (median of TLR4(1): 0.1330 vs. 0.1970, p < 0.0001 and median of TLR4(4): 0.1840 vs. 0.2066, p = 0.0353) (Figure 2A,C).There was no difference in the expression of TLR4(3) in CLL patients and HVs (0.1680 vs. 0.1775, p = 0.0592) (Figure 2B).The expression of TLR7 and TLR9 was significantly higher in CLL patients compared to HVs (0.4790 vs. 0.1877, p < 0.0001), (0.3735 vs. 0.1066, p < 0.0001), respectively (Figure 1B,C).0.2185 vs. 0.2632 (p = 0.039) (Figure 1A).Similarly, the expression of splicing variants of TLR( 4), including TLR4(1) and TLR4(4), was significantly lower in CLL patients than in HVs (median of TLR4(1): 0.1330 vs. 0.1970, p < 0.0001 and median of TLR4(4): 0.1840 vs. 0.2066, p = 0.0353) (Figure 2A,C).There was no difference in the expression of TLR4(3) in CLL patients and HVs (0.1680 vs. 0.1775, p = 0.0592) (Figure 2B).The expression of TLR7 and TLR9 was significantly higher in CLL patients compared to HVs (0.4790 vs. 0.1877, p < 0.0001), (0.3735 vs. 0.1066, p < 0.0001), respectively (Figure 1B,C).TLR( 4), including TLR4(1) and TLR4( 4), was significantly lower in CLL patients than in HVs (median of TLR4(1): 0.1330 vs. 0.1970, p < 0.0001 and median of TLR4( 4): 0.1840 vs. 0.2066, p = 0.0353) (Figure 2A,C).There was no difference in the expression of TLR4( 3) in CLL patients and HVs (0.1680 vs. 0.1775, p = 0.0592) (Figure 2B).The expression of TLR7 and TLR9 was significantly higher in CLL patients compared to HVs (0.4790 vs. 0.1877, p < 0.0001), (0.3735 vs. 0.1066, p < 0.0001), respectively (Figure 1B,C). ",
"section_name": "Aberrant Expression of TLR2, TLR7, TLR9 and Splicing Variants of TLR4 in CLL Patients Compared to Healthy Volunteers",
"section_num": "3.1."
},
{
"section_content": "To analyze a difference in the regulation of TLRs expression in biologically various compartments, we performed a comparison of the levels of expression of TLRs in peripheral blood and bone marrow mononuclear cells.There was no difference in TLR2, TLR7, and TLR9 expression in PBMC and BMMC with the median expression 0.2185 vs. 0.2 (p = 0.178); 0.479 vs. 0.4665 (p = 0.7215); 0.3735 vs. 0.368 (p = 0.8333), respectively.There was also no difference in expression of splicing variants of TLR4 including TLR4(1), TLR4(3), TLR4(4) in PBMC and BMMC (0.1330 vs. 0.1270, p = 0.8117), (0.1680 vs. 0.1520, p = 0.0952), (0.184 vs. 0.167, p = 0.0952). ",
"section_name": "Expression of TLR2, TLR7, TLR9 and Splicing Variants of TLR4 in Peripheral Blood and Bone Marrow Compartments in CLL",
"section_num": "3.2."
},
{
"section_content": "To characterize the significance of TLRs in CLL in a prognostic context we characterized the impact of TLRs expression on the clinical outcome and association with the recognized prognostic markers in both compartments, including PBMC and BMMC. We observed no difference in TTFT in subgroups of patients with a high and low level of the TLR2 expression in PBMC (p = 0.7229) as well as in BMMC (p = 0.4523). We found that the expression of TLR2 in BMMC was significantly higher in CLL patients with the unmutated status of the immunoglobulin heavy chain variable (UM IGHV) genes than in patients with IGHV mutation (MUT) with a median 0.212 vs. 0.1795 (p = 0.0181), respectively (Figure 3A). We found that the expression of TLR2 in BMMC was significantly higher in CLL patients with the unmutated status of the immunoglobulin heavy chain variable (UM IGHV) genes than in patients with IGHV mutation (MUT) with a median 0.212 vs. 0.1795 (p = 0.0181), respectively (Figure 3A). The expression of TLR2 in BMMC was confirmed to be higher in ZAP-70+ (defined as cytoplasmatic expression >20% CLL cells) patients compared to ZAP-70-, with a median 0.2225 vs. 0.1885 (p = 0.0014), respectively (Figure 3B).However, in PBMC there were no differences in the expression of TLR2 depending on ZAP-70 expression (median expression 0.215 vs. 0.222, p = 0.8538) as well as the mutational status of IGHV genes (median expression 0.225 vs. 0.204, p = 0.8690).The expression of TLR2 in BMMC was confirmed to be higher in ZAP-70+ (defined as cytoplasmatic expression >20% CLL cells) patients compared to ZAP-70-, with a median 0.2225 vs. 0.1885 (p = 0.0014), respectively (Figure 3B).However, in PBMC there were no differences in the expression of TLR2 depending on ZAP-70 expression (median expression 0.215 vs. 0.222, p = 0.8538) as well as the mutational status of IGHV genes (median expression 0.225 vs. 0.204, p = 0.8690). We found that there was no difference in TLR2 expression in PBMC (median expression 0.212 vs. 0.2185, p = 0.5737) and BMMC (median expression 0.2055 vs. 0.2, p = 0.9788) in the groups of patients defined by CD38 expression (cut-off value for the positive expression = 30%). Additionally, there were no statistical differences in TLR2 expression in stages A, B, and C according to Binet's classification (Table S1).We did not find any correlation between expression of TLR2 in PBMC and BMMC and CLL patient's age (r = -0.04755,p = 0.6582), (r = 0.05174, p = 0.6301), respectively. ",
"section_name": "Prognostic Value of the TLR2 Expression in Peripheral Blood and Bone Marrow Compartments in CLL",
"section_num": "3.3."
},
{
"section_content": "Analysis of survival curves found no difference in TTFT in subgroups of patients with a high and low level of the TLR7 expression in PBMC (p = 0.6819) as well as in BBMC (p = 0.5472). In the groups of CLL patients categorized by the mutational status of IGHV genes and ZAP-70 expression, we found that in PBMC there were no differences in TLR7 expression in IGHV MUT and IGHV UM CLL patients (0.4805 vs. 0.583, p > 0.05) as well as in ZAP-70and ZAP-70+ CLL patients (0.4935 vs. 0.532, p = 0.3738).Interestingly, in BMMC we found that expression of TLR7 was significantly higher in IGHV UM patients compared to IGHV MUT (0.549 vs. 0.407 vs., p < 0.0361) (Figure 3C) as well as in ZAP-70+ patients compared to ZAP-70-(0.694vs. 0.424 vs., p < 0.0001) (Figure 3D). There was no difference in TLR7 expression in CD-38+ and CD-38-patients in both peripheral blood and bone marrow compartments (0.668 vs. 0.4805, p = 4515 and 0.6895 vs. 0.4435, p = 0.0647), respectively.No association was observed in TLR7 expression in peripheral blood and bone marrow with the clinical stage of disease according to Binet's classification (Table S1). We did not find a correlation between the expression of TLR7 in peripheral blood and bone marrow samples and CLL patient's age (r = 0.1755, p = 0.1, r = 0.03711, p = 0.7299), respectively. ",
"section_name": "Prognostic Value of the TLR7 Expression in Peripheral Blood and Bone Marrow Compartments in CLL",
"section_num": "3.4."
},
{
"section_content": "We showed the tendency of shorter TTFT in groups of CLL patients with high TLR9 expression in comparison to low TLR9 expression in BBMC (12 vs. 45, p = 0.0655).In PBMC there was no difference in TTFT, referring to the level of TLR9 expression (p = 0.3210). There was no difference in the expression of TLR9 in peripheral blood as well as in bone marrow in IGHV MUT CLL patients mutated compared to IGHV UM (0.4345 vs. 0.367, p = 0.2104; median expression 0.3375 vs. 0.353, p = 0.6380, respectively). There was no difference in TLR9 expression between groups of patients characterized by the expression of ZAP-70 both in peripheral blood (0.3524 vs. 0.4190, p = 0.2327) and bone marrow samples (0.3375 vs. 0.407, p = 0.1038), respectively.In PBMC we found that expression of TLR9 was significantly higher in CD-38-compared to CD-38+ patients (0.4005 vs. 0.295, p = 0.0234).In contrast, in bone marrow samples we did not find a statistically significant difference in TLR9 expression between CD-38+ and CD-38patients (0.4025 vs. 0.3583, p = 0.6519). No differences were observed in TLR9 expression in peripheral blood between stages A, B, and C according to Binet's classification in PBMC and BMMC (Table S1).We did not find any correlation between the expression of TLR9 in peripheral blood and bone marrow samples and CLL patient's age (r = -0.01817,p = 0.8658: r = 0.07727, p = 0.4717), respectively. ",
"section_name": "Prognostic Value of the TLR9 Expression in Peripheral Blood and Bone Marrow Compartments in CLL",
"section_num": "3.5."
},
{
"section_content": "To define the prognostic value of TLR(4) splicing variants, we divided TTFT according to the level of the TLR4(1), TLR4(3), TLR4(4) expression in PBMC and BMMC.Analysis of survival curves found that TTFT was not different when referring to the level of TLR4(1) expression in PBMC (p = 0.5674) as well as BMMC (p = 0.3008).We found shorter TTFT in patients with low expression of TLR4(3) compared to patients with high expression of TLR4(3) in BBMC (13 vs. 48, p = 0.0207) (Figure 4A).A tendency to have shorter TTFT was found in patients with low expression of TLR4(4) compared to high expression in BBMC (10 vs. 48, p = 0.0828) (Figure 4B).However, in PBMC we did not observe changes in TTFT regarding TLR4(3) (p = 0.01761) and TLR4(4) expression. of survival curves found that TTFT was not different when referring to the level of TLR4(1) expression in PBMC (p = 0.5674) as well as BMMC (p = 0.3008).We found shorter TTFT in patients with low expression of TLR4(3) compared to patients with high expression of TLR4(3) in BBMC (13 vs. 48, p = 0.0207) (Figure 4A).A tendency to have shorter TTFT was found in patients with low expression of TLR4( 4) compared to high expression in BBMC (10 vs. 48, p = 0.0828) (Figure 4B).However, in PBMC we did not observe changes in TTFT regarding TLR4(3) (p = 0.01761) and TLR4(4) expression.3) expression compared to the group of patients with a high level of TLR4(3) expression in BMMC (13 vs. 48, p = 0.0207).(B) TTFT tended to be shorter in the group of patients with a low level of TLR4(4) expression compared to the group of patients with a high level of TLR4(4) expression in BMMC (10 vs. 48, p = 0.0828).(C) TTFT tended to be shorter in the group of patients with a high level of TLR9 expression compared to the group of patients with a low level of TLR9 expression in BMMC (12 vs. 45, p = 0.0655). We analyzed the expression of splicing variants of TLR4 (TLR4(1), TLR4(3), TLR4( 4)) with respect to the mutational status of IGHV genes.There were no differences in expression of TLR4 (1) in IGHV UM CLL cases compared to IGHV MUT (0.124 in vs. 0.141, p = 0.1316) in PBMC as well as in BMMC (0.1265 vs. 0.131, p = 0.493).We found that expression 3) expression compared to the group of patients with a high level of TLR4(3) expression in BMMC (13 vs. 48, p = 0.0207).(B) TTFT tended to be shorter in the group of patients with a low level of TLR4(4) expression compared to the group of patients with a high level of TLR4(4) expression in BMMC (10 vs. 48, p = 0.0828).(C) TTFT tended to be shorter in the group of patients with a high level of TLR9 expression compared to the group of patients with a low level of TLR9 expression in BMMC (12 vs. 45, p = 0.0655). We analyzed the expression of splicing variants of TLR4 (TLR4(1), TLR4(3), TLR4(4)) with respect to the mutational status of IGHV genes.There were no differences in expression of TLR4(1) in IGHV UM CLL cases compared to IGHV MUT (0.124 in vs. 0.141, p = 0.1316) in PBMC as well as in BMMC (0.1265 vs. 0.131, p = 0.493).We found that expression of TLR4(3) and TLR(4) in PBMC were lower in patients with IGHV UM compared to IGHV MUT (0.158 vs. 0.1815, p = 0.0233) (0.1695 vs. 0.2015 vs, p = 0.00642), respectively.The expression was found to be no different in BBMC in patients with IGHV MUT compared to IGHV UM (0.1585 vs. 0.137, p = 0.3188). The expressions of TLR4(1), TLR4(3) and TLR4(4) were analyzed in two groups of CLL patients: ZAP-70+ and ZAP-70-.The expression of TLR4(1) in PBMC was found to be lower in ZAP-70+ patients than in ZAP-70+ (median expression 0.122 vs. 0.139, p = 0.0305).In bone marrow samples we also did not find differences in expression of TLR4(1) in ZAP-70+ and ZAP-70-CLL patients (0.157 vs. 0.152, p = 0.7583).We evaluated the expression of TLR4(3) in those groups of CLL patients.We observed that there was no difference in expression of TLR4(3) in PBMC and BMMC in ZAP-70-and ZAP-70+ patients (0.172 vs. 0.159, p = 0.0541), (0.152 vs. 0.157, p = 0.7583), respectively.The expression of TLR4(4) was similar in ZAP-70-and ZAP-70+ patients (0.1965 vs. 0.173, p = 0.2039) in PBMC as well as in BMMC (0.17 vs. 0.168, p = 0.804). We determined the expression of splicing variants of TLR4 (TLR4(1), TLR4(3), TLR4(4)) in CD38+ and CD38-CLL patients.There were no differences in expression of TLR4(1) (0.126 vs. 0.137, p = 0.3911), TLR4(3) (0.16 vs. 0.171, p = 0.3079) and TLR4(4) (0.192 vs. 0.184, p = 0.5018) in CD-38+ and CD-38-groups in PBMC.In bone marrow, differences in expression of TLR4(1) (0.121 vs.0.13 p = 0.4011), TLR4(3) (0.156 vs. 0.152, p = 0.6144), TLR4(4) (0.176 vs. 0.168, p = 0.679) in CD38+ and CD38-CLL patients were not also observed. We did not find any difference in expression of splicing variants of TLR4 in stages A, B, and C according to Binet's classification in PBMC and BMMC (Table S1).We did not find correlation between expression of splicing variants of TLR4 in peripheral blood (TLR4(1): r = -0.0705,p = 0.5188, TLR4(3): r = -0.1203,p = 0.2698, TLR4(4): r = 0.8704) and bone marrow samples TLR4(1):r = -0.1613,p = 0.1378, TLR4(3): r = -0.04505,p = 0.6804, TLR4(4): r = -0.02286,p = 0.8345) and CLL patient's age. ",
"section_name": "Prognostic Value of the Expression of Splicing Variants of TLR4 (TLR4(1), TLR4(3), TLR4(4)) in Peripheral Blood and Bone Marrow Compartments in CLL",
"section_num": "3.6."
},
{
"section_content": "The MYD88 expression was higher in CLL patients compared to HVs with a median of 0.1780 vs. 0.128 (p < 0.0001), respectively.The median expression of MYD88 in BMMC was 0.1600 (Figure 5A).We revealed no differences in TTFT in subgroups of patients with high and low expression of MYD88 in bone marrow (10 vs. 26, p = 0.92) as well as in peripheral blood (11 vs. 21, p = 0.59).To identify association of MYD88 expression and TLRs expression, we assessed correlations in blood and bone marrow samples (Table 3).To identify association of MYD88 expression and TLRs expression, we assessed correlations in blood and bone marrow samples (Table 3).We showed a strong correlation between MYD88 expression and TLR2 in PBMC (r = 0.722, p ≤ 0.0001) and medium correlation in BMMC (r = 0.389, p = 0.0001) (Figure S1A,B).There were medium correlations between expression of MYD88 and expression of TLR4 splicing variants including TLR4(1) (r = 0.559, p < 0.001), TLR(3) (r = 0.558, p < 0.001), and TLR4(4) (r = 0.56, p < 0.001) in PBMC (Figure S2A,C,E).In BMMC there were weak associations between expression of MYD88 and expression of TLR4(1) (r = 0.284, p < 0.007), TLR(3) (r = 0.294, p < 0.005), TLR4(4) (r = 0.44, p < 0.001) (Figure S2B,D,F).We observed medium correlation between MYD88 expression and TLR7 expression in PBMC (r = 0.580, p < 0.001) and medium correlation in BMMC (r = 0.358, p < 0.001) (Figure S3A,B) There were medium correlations between MYD88 expression and TLR9 expression in PBMC (r = 0.492, p < 0.001) as well as in BMMC (r = 0.541, p < 0.001) (Figure S4A,B). To determine the significance of MYD88 in a different compartment we performed statistical analyses referring to CLL subgroups of patients with different prognoses in blood as well as bone marrow samples.We observed no association of expression of MYD88 with clinical stage of CLL according to Binet's scale in PBMC with the median expression in A, B, C Binet's stage: 0.1765 vs. 0.1605 vs. 0.2070, p = 0.4868, respectively (Figure 5B).However, BMMC expression of MYD88 was found to be higher in A Binet stage compared to B and C Binet stages in BMMC (0.1720 vs. 0.1535 vs. 0.1550, p < 0.0001) (Figure 5C). No correlation between MYD88 expression in blood and bone marrow samples in CLL patients (r = 0.04, p = 0.715) was found. We analyzed if the expression of MYD88 in blood and bone marrow samples in CLL patients depends on the prognostic markers including the mutational status of IGHV genes, ZAP-70 expression, CD38 expression, and Binet stage.We observed no differences in the expression of MYD88 in IGHV MUT patients compared to IGHV UM in PBMC with a median 0.1831 vs. 0.1926 (p = 0.4934), respectively, as well as in BMMC with a median 0.1603 vs. 0.1605 (p = 0.8730), respectively.There were no differences in the expression of MYD88 in ZAP-70+ CLL patients compared to ZAP-70-with a median 0.1734 vs. 0.1816 (p = 0.6426) in PBMC and BMMC with a median 0.1646 vs. 0.1601 (p = 0.9589).There were also no differences in the expression of MYD88 in CD38+ CLL patients compared to CD38in PBMC with a median 0.2112 vs. 0.1778 (p = 0.867), respectively, as well as in BMMC with a median 0.1553 vs.0.1636 (p = 0.1855), respectively. There were no correlations between MYD88 expression and the age of CLL patients in peripheral blood (p = 0.7874) and bone marrow (p = 0.3189) samples.We did not find any associations between MYD88 expression and morphological parameters, including the level of white blood cells (WBC) (p = 0.7873), red blood cells (RBC) (p = 0.537), platelets (PLT) (p = 0.2014), neutrophils (p = 0.981), and the level of hemoglobin (Hgb) (p = 0.570) and hematocrit (Hct) (p = 0.5017) in blood samples.There were no correlations between MYD88 expression and morphological parameters, including the level of white blood cells (WBC) (p = 0.9314), red blood cells (RBC) (p = 0.1792), platelets (PLT) (p = 0.2209), neutrophils (0.1007), and the level of hemoglobin (Hgb) (p = 0.3833) and hematocrit (Hct) (p = 0.3389) in bone marrow. ",
"section_name": "Prognostic Value of the MYD88 Expression and the Association with TLRs Expression in CLL",
"section_num": "3.7."
},
{
"section_content": "MYD88 L265P mutation occurred in 2/61 (3.28%) CLL patients in PBMC.Since a small number of the cohort had a MYD88 mutation, we did not aim to obtain median and statistical data.We obtained mean values.In patients with MYD88 L265P mutation, the mean expression of splicing variants of TLR4 (1/∆Ct) was as follows: TLR4(1)-0.165,TLR4(3)-0.153,TLR4(4)-0.138.In the group of patients without MYD88 L265P mutation, the median expression of splicing variants of TLR4 (1/∆Ct) was 0.138 for TLR4(1), 0.171 for TLR4(3), and 0.184 for TLR4(4).In the population of MYD88-mutated patients, the mean expression of TLR2 (1/∆Ct) was 0.203, meanwhile patients without MYD88 mutation had a median of 0.225.The mean expression of TLR7 (1/∆Ct) in patients with MYD88 L265P mutation was 0.866, and in the case of patients without MYD88 L265P mutation, the median expression of TLR7 (1/∆Ct) was 0.573.In patients harboring the MYD88 L265P mutation, the mean expression of TLR9 (1/∆Ct) was 0.517, meanwhile patients without MYD88 L265P mutation had a median of 0.405. ",
"section_name": "MYD88 L265P Mutation in CLL Patients",
"section_num": "3.8."
},
{
"section_content": "In tumors, functional TLRs expression could influence preferable anti-tumor effects by activating inflammatory cytokine expression and cytotoxic T-cells response.However, an excessive TLRs activation could promote tumor progression, since TLR-induced inflammation stimulates cancer cells boost in the microenvironment [16].Several reports demonstrated changes of immunological parameters between accumulation and proliferation compartments of the microenvironment in CLL [19,20].There are some studies that suggest differential signaling trough BCR in an accumulative and proliferative compartment in CLL microenvironment and provide their differential involvement in biology and pathogenesis in this disease [19,21].It was found that especially in the lymph node microenvironment of CLL, both BCR and TLR signaling could contribute to NF-κB activation, which is essential in immune response regulation, oncogenesis, and tumor progression [21].So far there are many questions about the expression pattern of TLRs and their prognostic significance in CLL, especially in the bone marrow microenvironment [12,22,23]. Results of our work showed aberrant expression of TLRs in CLL patients compared to HVs, proving that TLRs expression is differentially regulated in CLL.We showed higher expression of TLR7 and TLR9 in CLL compared to HVs, which was confirmed in earlier reports [24,25].Arvaniti et al. [11] demonstrated high expression of TLR7, while the expression of TLR9 was showed to be low on a mRNA level.Other studies showed variable levels of mRNA and low protein expression of TLR9 [26].Chatzouli et al. [27] reported that TLR7 and TLR9 stimulation with agonists results in apoptosis of CLL cells but only in IGHV mutated patients.Recent studies provided by Zhao et al. [28] demonstrated that phenotypically identical cells nevertheless express very different levels of each receptor.The authors suggested that the engagement of multiple receptors, such as TLR7/8/9, may be necessary for improving the host anti-tumor response.Activation of TLR7/8/9 leads to antigen-specific humoral responses through B-lymphocyte activation, but also the inhibition of B-cell apoptosis.In contrast to higher TLR7 and TLR9 expression in CLL, expression of TLR2 and TLR4 was confirmed to be lower in CLL compared to HVs.Our results are in line with previous studies [7,[23][24][25].The expression of TLR2 and TLR7 was correlated with negative known prognostic markers, but only in the bone marrow compartment.Expanded expression analysis of splicing variants of TLR4 was assessed in CLL patients for the first time.Here, we showed that the expression of splicing variants of TLR4 (TLR4(1) and TLR4(4)) were significantly lower in PBMC in CLL compared to HV.The decreased expression of splicing variants of TLR4 observed in this study might then be a result of an impaired host response in CLL patients.Several TLR agonists have been used in clinical trials of CLL patients as adjuvants to improve the efficacy of chemotherapy, for example, the agonist of TLR4.Different studies demonstrate positive as well as negative effects of TLR4 stimulation on cancer development or treatment.Hwang et al. [29] indicated that stimulation of TLR4 results in IL-6, IL-8, IL-12, TNF, INF-γ, and CCL5 secretion.Stimulation of TLR4 that can enhance the anti-tumor response and the beneficial effects of TLR4 stimulation while eliminating the negative effects remains a challenge for cancer researchers. Signal transduction through TLRs is involved in B cell biology, including activation of naïve B cells, differentiation, and induction proliferation of memory B cells.Expression of TLR is associated with their ability to respond to TLR agonists.Naïve B cells are characterized by low expression of TLR7 and TLR9, as well as TLR1, TLR6, TLR10, while memory B cells express a high level of TLR7 and TLR9, accompanied by a low level of TLR2, TLR4, TLR8 [11,30].Comparing our defined TLR profile of CLL cells to normal B cells, we can indicate that it is parallel to memory B cells.This analogy is in line with the previous study [30]. No differences in TLRs expression between peripheral blood and bone marrow indicate similarities shared between these two distinct compartments of the CLL microenvironment.However, we suggest that differential regulation of TLRs accompanies its variable prognostic value, depending on the microenvironment of the specific compartment of CLL.Interestingly, our results for the first time showed the impact of splicing variants of TLR4(3) on clinical outcomes in CLL.Analysis of survival curves revealed that TTFT was significantly shorter in the group of patients with a low level of TLR4(3) expression compared to the group of patients with a high level of TLR4(3) expression in BMMC, thereby we could indicate the negative prognostic value of low TLR4(3) expression assessed in the bone marrow compartment in CLL.Moreover, our results might indicate also a potential negative prognostic value of the low level of TLR4(4) in bone marrow since expression of TTFT tended to be shorter in the group of patients with a low level of TLR4(4) expression compared to the group of patients with a high level of TLR4(4) expression in BMMC.Analogous results were not obtained in peripheral blood, as well as in terms of TLR4 (1).Thereby, we might indicate that deregulation of TLR4 signaling via TLR4(3) and TLR( 4) is more relevant in bone marrow as a proliferative than accumulative compartment.These differences might be explained by differential posttranscriptional regulation of TLR4(1), TLR4(3) and TLR(4) and diverse functional and cellular interactions in those two compartments of CLL.Although TLR4 splicing variants differ in terms of the number of exons and length of the extracellular domain, their functional importance in a cell has not been analyzed [31,32].The significance of TLR4 in disease progression was shown also in human lung cancers [33], as well as ovarian cancer [34].In mantle cell lymphoma (MCL), Wang et al. [35] showed that signaling through TLR4 triggers a cascade leading to the growth of cells and evasion from immune surveillance, contributing to disease progression.However, Nunez et al. [36] showed that TLR4-activated tumor cells are engaged in an antitumoral immune response.This discrepancy might be associated with the type of cytokines that are secreted upon stimulation in a specific tumor microenvironment. Results of our study revealed a tendency for shorter TTFT in groups of CLL patients with high TLR9 expression in comparison to low TLR9 expression in BBMC, indicating the potential prognostic significance of high mRNA TLR9 expression in bone marrow in CLL.In PBMC, there was no difference in TTFT when referring to the level of TLR9 expression, while the results of our previous study [22] revealed that high expression of TLR9 on the protein level in CLL patients is correlated with longer TTFT.Interestingly, significant discrepancies were identified between mRNA and protein levels for certain TLR expression, and a high expression on a mRNA level did not always correspond to strong protein expression.Several factors could be responsible for these discrepancies, such as cellular intraclonal heterogeneity, differential activation status of malignant cells, and different cell viability in different samples.The expression of TLR2 and TLR7 was correlated with known negative prognostic markers but only in the bone marrow compartment. Since downstream signaling pathways through TLRs involve Myd88 as an adaptor molecule, we provided MYD88 expression patterns in peripheral blood and bone marrow, referring to CLL prognostic factors as well as an association between MYD88 and TLRs expression in both compartments.In the literature, there is only data about the prognostic significance of MYD88 mutation [17] or MYD88 expression.We revealed higher MYD88 expression in CLL patients compared to HVs, although Antosz et al. [37] revealed lower expression of MYD88 on a mRNA level in CLL compared to control.They found that TLR agonist stimulation did not result in changes in Myd88 protein expression and suggested some defects in Myd88 proteins in CLL.Myd88 involvement by acting downstream of TLRs in carcinogenesis was shown in many reports concerning cancer of the skin, pancreas, liver, colon, sarcoma [38], whereas the data about the mRNA role of MYD88 expression in tumors are limited.Chen et al. [39] showed that MYD88 as well as TLR4 mRNA expression was higher in breast cancer tissue compared to adjacent normal tissue.Moreover, subsequent research showed a higher protein level of Myd88 and TLR4 in breast carcinoma than paracarcinoma tissue, as well as a correlation between their expression and axillary lymph node metastasis that providing the metastatic potential role of TLR4/Myd88 signaling in breast cancer [40].However, in ovarian cancer it was also revealed that Myd88 expression strongly correlated with TRL4 expression and provided a favorable prognosis [41]. All TLRs expressions that were analyzed (TLR2, TLR7, TLR9, and TLR4 isoforms) represent components of downstream cell signaling pathways by Myd88 that finally activate NF-κB, which is essential for CLL survival.It has been proven that TLRs, including 7 and 9, initiate the Myd88-dependent pathway directly, while TLRs types 2 and 4 indirectly impact the involvement of TIRAP.Specifically, TLR4 initiates an alternative pathway which is MyD88-independent by recruiting TRIF that eventually activates NF-κB. These mutual associations indicate their relevant cooperation which points to the utilization of Myd88 by TLRs signaling in CLL cells.Since the MyD88-dependent signaling pathway leads to the early phase of NF-κB activation whereas the MyD88-independent signaling pathway initiates the late phase of NF-κB activation, this suggests that both mechanisms might be utilized in CLL. To sum up, our results proved correlations between MYD88 and analyzed TLRs expressions in both compartments, indicating their relevant cooperation in signal transduction in CLL cells.The MYD88 expression was higher in CLL patients compared to HVs.The TLRs expression was aberrant in CLL patients compared to HVs.Differences in TTFT indicate a negative prognostic value of low TLR4(3) expression in the bone marrow, which might suggest an importance of deregulation of the signaling pathway typical for TLR(4) (Myd88-independent/TRIF-dependent NF-κB activation) in this compartment. We proved that TLRs expression is differentially regulated in CLL but is similarly shared between two distinct compartments of the CLL microenvironment.We indicate that differential regulation of TLRs might accompany the various role of TLRs signaling in peripheral blood and bone marrow, representing two different microenvironmental compartments of CLL. ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "The following are available online at https://www.mdpi.com/2077-0383/10/4/867/s1, Figure S1: 637 Correlations between TLR2 expression and MYD88 expression in PBMC (A) and BMMC (B) in CLL., Figure S2: 638 Correlations between TLR4 splice variants expression and MYD88 expression in PBMC (A, C, E), and BMMC (B,D,F) in CLL., Figure S3: 639 Correlations between TLR7 expression and MYD88 expression in PBMC (A) and BMMC (B) in CLL., Figure S4: 640 Correlations between TLR9 expression and MYD88 expression in PBMC (A) and BMMC (B) in CLL., Table S1.Expression of TLRs in A, B, C clinical stage of disease according to Binet scale. ",
"section_name": "Supplementary Materials:",
"section_num": null
}
] |
[
{
"section_content": "The authors would like to thank to Magdalena Osiak, Natalia Pajak, Malgorzata Zajac, Joanna Zaleska, Marta Karp, Maciej Grzywnowicz for laboratory support and Waldemar Tomczak for patient recruitment and providing clinical data. ",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "The data presented in this study are available on request from the corresponding author. ",
"section_name": "Data Availability Statement:",
"section_num": null
},
{
"section_content": "Author Contributions: Conceptualization, K.G.; methodology, K.S., A.K.; software, K.S., A.K.; validation, K.S., P.W. and K.G.; formal analysis, K.S.; investigation, K.S., A.K.; resources, K.G.; data curation, K.S., A.K., P.W.; writing-original draft preparation, K.S., P.W.; writing-review and editing, K.G., K.S.; visualization, K.S.; supervision, K.G.; project administration, K.G.; funding acquisition, K.G.K.G. designed the research.K.S., A.K. performed the research.K.S., P.W. and A.K. analyzed data.K.S. and P.W. wrote the paper.K.G., K.S. and P.W. discussed results.All authors have read and agreed to the published version of the manuscript.Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. The authors declare no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "Author Contributions: Conceptualization, K.G.; methodology, K.S., A.K.; software, K.S., A.K.; validation, K.S., P.W. and K.G.; formal analysis, K.S.; investigation, K.S., A.K.; resources, K.G.; data curation, K.S., A.K., P.W.; writing-original draft preparation, K.S., P.W.; writing-review and editing, K.G., K.S.; visualization, K.S.; supervision, K.G.; project administration, K.G.; funding acquisition, K.G.K.G. designed the research.K.S., A.K. performed the research.K.S., P.W. and A.K. analyzed data.K.S. and P.W. wrote the paper.K.G., K.S. and P.W. discussed results.All authors have read and agreed to the published version of the manuscript.Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors declare no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
}
] |
10.1007/s10238-015-0360-7
|
CTLA-4 affects expression of key cell cycle regulators of G0/G1 phase in neoplastic lymphocytes from patients with chronic lymphocytic leukaemia
|
Previously, we showed that cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is overexpressed in chronic lymphocytic leukaemia (CLL) and its expression is correlated with the expression of the major regulators of G1 phase progression: cyclins D2 and D3, and cyclin-dependent kinase inhibitory protein 1 (p27 (KIP1) ). In the present study, we blocked CTLA-4 on the surface of both CLL cells and normal B lymphocytes to investigate the impact of CTLA-4 on the expression of the mentioned G1 phase regulators. We found that in CLL patients and in healthy individuals, the median proportions of cyclin D2-positive cells as well as cyclin D3(+) cells significantly decreased following CTLA-4 blockade. Moreover, CTLA-4 blockade led to an increase in the median frequencies of p27 (KIP1) -positive cells, although this increase was marked only in CLL patients. Our study showed that CTLA-4 affects the expression of the key regulators of G1 phase progression in CLL cells as well as in normal B lymphocytes and may contribute to a better understanding of the role of CTLA-4 in the regulation of G1 phase progression.
|
[
{
"section_content": "Chronic lymphocytic leukaemia (CLL) is the most common form of leukaemia in adults in Western Europe and North America.It has been described as a progressive accumulation of malignant, morphologically mature CD19 ?CD5 ?cells in the peripheral blood, bone marrow, and lymphoid organs [1][2][3].The disease is characterised by a highly variable clinical presentation and evolution [4,5].Some patients exhibit stable disease over years and never require a therapeutic intervention, whereas others progress rapidly towards more advanced stages and succumb relatively soon despite aggressive treatment.Based on genetic, phenotypic, and molecular characteristics of CLL, several prognostic markers have emerged in the past decade [6,7].It has been well documented that the highrisk phenotype is typically associated with unmutated immunoglobulin heavy variable genes (IgV H ) [8], high expression of the CD38 surface marker [9], or the f-chainassociated protein 70 kDa (ZAP-70) [10], as well as with chromosomal aberrations such as 17p (the site of tumour protein p53) or 11q23 deletions (the site of ataxia telangiectasia mutated ATM) [11].Recently, it has been postulated that the difference in clinical course among CLL patients may, at least in part, be determined by the proliferative capacity of CLL cells [12,13].It is becoming increasingly evident that the cells located in the proliferation centres (PCs) of lymph nodes and bone marrow proliferate more than previously anticipated [13], and CLL patients with higher birth rates are much more likely to exhibit active or to develop progressive disease than those with lower birth rates [13].Moreover, the proliferating cells in PCs represent the CLL proliferating reservoir that replenishes the downstream accumulation compartment. In contrast to the proliferating cells in PCs, the vast majority of CLL cells circulating in the peripheral blood are arrested in the G0/early G1 phase of the cell cycle.The key regulators of G1 phase progression in human T and B lymphocytes are the D-type cyclins cyclin D2 and D3, and p27 KIP1 (cyclin-dependent kinase inhibitory protein 1).The D-type cyclins positively regulate passage through the G1 phase by binding to and stimulating the activities of their catalytic partners, the cyclin-dependent kinases (CDKs) cdk4 and cdk6 [14][15][16][17], while p27 KIP1 exerts an inhibitory effect on the kinases cdk4 and cdk6 or their complexes with cyclin D2 or cyclin D3 [17][18][19].Moreover, one of the factors involved in regulation of cell cycle progression of T cells is cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4; CD152) [20,21].It has been well documented that CTLA-4 prolongs the progression of T cells through the G1 phase by influencing the expression of the major regulators of this cell cycle phase [20,21].CTLA-4 upregulates the expression of cyclin D2 and inhibits cyclin D3, cdk4, and cdk6 production in these cells.Furthermore, CTLA-4 affects the degradation of p27 KIP1 protein and contributes to its earlier and stronger re-expression during the late stages of T cell activation [20,21].In contrast to the well-documented involvement of CTLA-4 in the regulation of cell cycle progression in T cells [20,21], only limited information is known about the role of this protein in cell cycle progression in normal B cells and malignant B lymphocytes.Our previous study indicated that CTLA-4 is overexpressed in freshly drawn CLL cells and it may be involved in the regulation of G1 phase progression in these cells [22].We found that CTLA-4 expression positively correlated with both cyclin D2 and p27 KIP1 expression and negatively with cyclin D3 level.Moreover, CTLA-4 expression positively correlated with the percentage of leukaemic cells in G0/G1 phase.Here, we have extended our previous study to examine whether stimulation with DSP30, a CpG oligodeoxynucleotide (ODN), and rIL-2 influences CTLA-4 expression in CLL cells.The main aim of this study was to investigate whether the CTLA-4 molecule affects the expression of cell cycle regulators of G0/G1 phase.For that purpose, we blocked CTLA-4 on the surface of CLL cells using monoclonal anti-CTLA-4 antibodies to assess the expression of cyclins D2 and D3, and p27 KIP1 protein.To the best of our knowledge, such studies are lacking so far. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and methods",
"section_num": null
},
{
"section_content": "The study design was approved by the local Bioethical Committee at the Medical University of Wroclaw, Poland, and is in accordance with the Helsinki Declaration of 1975.All participants gave written informed consent after the purpose of the study was explained to them.Thirty-eight previously untreated CLL patients of the Clinic of Haematology, Blood Neoplasms, and Bone Marrow Transplantation, Wroclaw Medical University, Poland, were enrolled in this study.In each of them, the diagnosis was established according to generally accepted criteria including absolute peripheral blood lymphocytosis C5 9 10 9 /L and the co-expression of CD5, CD19, and CD23 antigens on malignant cells.The disease stages were determined according to the Rai classification.Clinical and laboratory features are presented in Table 1. Leucocyte-enriched fractions of peripheral blood donated by 15 healthy volunteers matched for age and sex with the CLL patients were purchased from the Regional Centre of Blood Donation and Treatment in Wroclaw, Poland. ",
"section_name": "Patients and healthy donors",
"section_num": null
},
{
"section_content": "Peripheral blood mononuclear cells (PBMCs) were separated from heparinised freshly drawn peripheral venous blood of CLL patients and healthy controls by buoyant density-gradient centrifugation on Lymphoflot (Bio-Rad Medical Diagnostics GmbH, Dreieich, Germany) and washed three times in phosphate-buffered saline (PBS) (without Ca 2? and Mg 2? ).The PBMCs were suspended in 95 % foetal calf serum (CytoGen GmbH, Sinn, Germany) For age and clinical parameters, the mean values and standard deviation (SD) were presented containing 5 % DMSO (Sigma-Aldrich, St. Gallen, Switzerland) and stored in liquid nitrogen until used.CLL cells were isolated from PBMCs by negative selection using EasySep Human B Cell Enrichment Kit without CD43 Depletion (STEMCELL Technologies Inc, Vancouver, Canada) according to the manufacturer's instructions.Following this separation procedure, more than 98 % of the resulting cell population was CD19 ?CD5 ? as assessed by flow cytometry using anti-CD19 and anti-CD5 monoclonal antibodies (mAbs) (Becton-Dickinson, BD Biosciences, San Diego, USA).Normal B cells from healthy individuals were isolated from PBMCs by negative selection using EasySep Human B Cell Enrichment Kit (STEMCELL Technologies Inc, Vancouver, Canada) according to the manufacturer's instructions, achieving above 98 % purity as assessed by flow cytometry using anti-CD19 mAb. ",
"section_name": "Cell isolation and separation procedures",
"section_num": null
},
{
"section_content": "Purified normal CD19 ?lymphocytes or CLL cells were suspended at 1 9 10 6 cells/ml in RPMI-1640 medium (Gibco, Paisley, UK) supplemented with 10 % foetal calf serum (CytoGen GmbH, Sinn, Germany), 2 mmol/l L-g- lutamine and 50 lg/ml gentamycin (KRKA-Poland, Warsaw, Poland), and cultured using 24-well U-bottom culture plates (Nunc GmbH & Co. KG, Langenselbold, Germany) at 37 °C in a 5 % CO 2 humidified atmosphere for 24 and 72 h either in medium alone or together with 1 lM DSP30 (5 0 -TCGTCGCTGTCTCCGCTTCTTCTTGCC-3 0 ) (TIB MOLBIOL, Berlin, Germany) [23] and 100 U/ml rIL-2 (Eurocetus, Amsterdam, The Netherlands) [24].For the blocking experiment, purified CLL cells and normal CD19 ?lymphocytes were cultured with 1 lM DSP30 and 100 U/ml IL-2 with the blocking anti-CTLA-4 mAbs (50 lg/ml) (BD Pharmingen, BD Biosciences, San Diego, USA) [25] or control IgG2 (50 lg/ml) (BD Pharmingen, BD Biosciences, San Diego, USA). ",
"section_name": "Culture conditions",
"section_num": null
},
{
"section_content": "The expression of these molecules was studied in purified CLL cells and normal CD19 ?lymphocytes before and after 24 and 72 h culture by a single immunostaining method. Briefly, for detection of surface expression of the CTLA-4 molecule, the cells were washed twice in PBS (without Ca 2? and Mg 2? ), divided into tubes at a concentration of 5 9 10 5 cells per tube and incubated with anti-CTLA-4 (CD152)/RPE mAbs (BD Pharmingen, BD Biosciences, San Diego, USA) for 30 min at 4 °C in the dark.Excess unbound antibodies were removed by two washes with PBS.Following these washes, the cells were resuspended in PBS and analysed by flow cytometry using a FACSCalibur flow cytometer (Becton-Dickinson, BD Biosciences, San Diego, USA).For determination of intracellular CTLA-4 expression, the cells were first fixed for 10 min at room temperature in 2 % paraformaldehyde (Fluka, Sigma-Aldrich, Buchs, Germany), washed in PBS, and incubated for 10 min at room temperature in BD Permeabilizing Solution 2 (Becton-Dickinson, BD Biosciences, San Diego, USA) according to the manufacturer's instructions.Then, the cells were incubated with anti-CTLA-4 (CD152)/RPE mAbs for 30 min at 37 °C in the dark. For detection of cyclins D2 and D3, and p27 KIP1 protein, the cells were fixed, permeabilised, and stained with anticyclin D2/FITC, anti-p27 KIP1 /FITC mAbs (Santa Cruz Biotechnology, Inc, Heidelberg, Germany), and anti-cyclin D3/FITC mAb (BD Pharmingen, BD Biosciences, San Diego, USA) according to the manufacturer's instructions. Negative controls were always done by omitting the mAbs and by incubating the cells with mouse Ig of the same isotype as the mAbs conjugated with RPE or FITC.At least, 10,000 events per sample were analysed.The results were expressed as the proportion of CTLA-4-, cyclin D2-, cyclin D3-, or p27 KIP1 -positive cells.The CellQuest program was used for statistical analysis of the acquired data. ",
"section_name": "Immunostaining of CTLA-4 and cell cycle regulators of G0/G1 phase, and flow cytometric analysis",
"section_num": null
},
{
"section_content": "Statistical analyses of the clinical data and laboratory findings were conducted using Statistica 10.0 or PQStat software.For clinical parameters, the mean values and standard deviation were calculated.For all other analysed variables, the median values and 25th and 75th interquartile range were calculated.All collected data were examined for normal distribution and for homogeneity of variances using the Shapiro-Wilk test and Levene's test, respectively.If data were normally distributed and had homogeneous variances, the comparisons between the studied groups were performed using the Student t test for independent samples.If data were not normally distributed and/or had heterogeneous variances, the nonparametric Mann-Whitney U test was used.To test the effects of culture and CTLA-4 blockade on analysed variables, the repeated measures ANOVA and the Student t test for dependent samples were used.If data were not normally distributed and/or had heterogeneous variances, the Friedman ANOVA test followed by a post hoc test (Dunn test), and the nonparametric Wilcoxon signed-rank test were applied.The relationship between the clinical parameters and CTLA-4 expression was tested with Spearman' rank correlation coefficient.In all analyses, differences were considered significant when P B 0.05. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Since CTLA-4 is transiently expressed on the cell surface and is predominantly located in intracellular compartments due to constitutive internalisation from the plasma membrane [26], we determined both surface and intracellular expression of this molecule.Similarly, to our earlier study [22], we observed significantly higher median proportions of leukaemic cells co-expressing the CTLA-4 molecule on the surface (sCTLA-4) as well as in cytoplasmic compartments (cCTLA-4) in CLL patients compared to the median percentages of the corresponding cells in healthy individuals (Table 2).Moreover, as we previously showed [22], the frequencies of both the sCTLA-4-positive and cCTLA-4-positive CLL cells were very variable in the studied patients, ranging from 3.5 to 57.1 % and from 10.1 to 74.1 %, respectively.Furthermore, we found negative correlations between sCTLA-4 as well as cCTLA-4 expression in leukaemic cells and the following clinical parameters: Rai stage (r = -0.46,P = 0.01 and r = -0.43,P = 0.02), leucocyte count (r = -0.48,P = 0.009 and r = -0.66,P = 0.0002), and lymphocyte count (r = -0.45,P = 0.02 and r = -0.64,P = 0.0003) (Fig. 1). As regards cell cycle regulators of G0/G1 phase, we observed significantly higher median proportions of both cyclin D2-positive and p27 KIP1 -positive cells in the CLL group compared to the median percentages of the corresponding cells in healthy volunteers (Table 3).In contrast, the median frequency of cyclin D3 ?cells in CLL patients was markedly lower compared to healthy controls (Table 3). Expression of CTLA-4 molecule in DSP301rIL-2stimulated CLL lymphocytes and normal CD19 1 cells We found a significant impact of the cell culture in medium alone as well as ex vivo stimulation with DSP30?rIL-2 on the surface and intracellular expression of the CTLA-4 molecule in both CLL patients and healthy individuals.As regards surface expression, in CLL patients as well as in healthy individuals, a marked decrease in the median proportions of sCTLA-4-positive cells after 72 h of cell culture in medium alone was found (Table 2; Figs.2,3).Consequently, the median percentage of sCTLA-4 ?cells in CLL patients remained higher than in healthy volunteers only after 24 h of culture (Table 2).In CLL patients, ex vivo stimulation led to a significant decrease in the median percentage of sCTLA-4 ?cells after 24 h of culture (Table 2; Figs.2,3).In contrast, in healthy volunteers, stimulation with DSP30?rIL-2 resulted in an increase in the median frequencies of sCTLA-4-positive lymphocytes compared to the control culture, although the differences were statistically significant only after 72 h of culture (Table 2; Figs.2,3).Moreover, no significant differences in the median proportions of sCTLA-4 ?cells between CLL patients and healthy individuals following ex vivo stimulation were found (Table 2). As regards cCTLA-4 expression, in CLL patients as well as in healthy individuals, a significant decrease in the median proportions of cCTLA-4-positive cells after 72 h of cell culture in medium alone was found (Table 2; Figs.2,4).Moreover, after 72 h of ex vivo stimulation, the median percentages of cCTLA-4 ?cells in both studied groups were significantly higher compared to the control culture (Table 2; Figs.2,4).Of note, the median proportions of cCTLA-4-positive cells in CLL patients remained higher than in healthy controls at each time point tested (Table 2).Simultaneously with CTLA-4 expression, we analysed the expression of cell cycle regulators of G0/G1 phase.As regards cyclin D2 expression, culture in medium alone resulted in a marked decrease in the median percentage of cyclin D2-positive cells after 72 h of culture in CLL patients (Table 3; Fig. 5).In contrast, in healthy individuals, the median proportions of CD19 ?cyclin D2 ?lymphocytes maintained stable during the control culture (Fig. 5).In consequence, the median frequency of cyclin D2-positive cells in CLL patients remained significantly higher than in healthy controls only after 24 h of control culture (Table 3).Moreover, in CLL patients, ex vivo stimulation led to a significant decline in the median proportion of the cyclin D2 ?cells after 72 h of cell culture compared to the value after 24 h of culture (Fig. 5).In contrast, in healthy individuals, after 24 h of ex vivo stimulation, we observed a marked increase in the median percentage of CD19 ?cyclin D2 ?cells (Fig. 5).Moreover, after 72 h of ex vivo stimulation, the median proportion of cyclin D2-positive cells markedly decreased compared to the value after 24 h of culture (Fig. 5).Furthermore, no significant differences in the median percentages of cyclin D2 ?cells between CLL patients and healthy individuals following ex vivo stimulation were found (Table 3). As regards cyclin D3 expression, culture in medium alone resulted in a marked decrease in the median percentage of cyclin D3-positive cells after 72 h in CLL patients as well as in healthy volunteers (Table 3; Fig. 5). Fig. 3 Representative examples of flow cytometric analyses of surface expression of CTLA-4 (sCTLA-4) on leukaemic cells (a) and normal B lymphocytes (b) before and after cell culture in medium alone and under stimulating conditions (DSP30?rIL-2).Numbers on dot plots represent the percentage of leukaemic or normal B lymphocytes expressing CTLA-4 on the cell surface Moreover, the median percentage of cyclin D3 ?cells in CLL patients remained markedly lower than in healthy volunteers only after 24 h of control culture (Table 3).In both studied groups, after 24 h of ex vivo stimulation, we observed a significant increase in the median proportions of cyclin D3 ?cells (Fig. 5).Moreover, in CLL patients as well as in healthy individuals, the median frequencies of cyclin D3-positive cells decreased markedly after 72 h of stimulating culture compared to the corresponding cells after 24 h of ex vivo stimulation (Fig. 5).Furthermore, in CLL patients, the median proportions of cyclin D3-positive cells were markedly lower compared with healthy volunteers under stimulation conditions. As regards p27 KIP1 expression, in both studied groups, no significant impact of the culture in medium alone on the expression of this protein was found (Table 3; Fig. 5).In CLL patients, ex vivo stimulation led to a gradual decrease in the median frequencies of p27 KIP1 -positive cells with the minimum values after 72 h of culture.Likewise, in healthy individuals, the median percentages of CD19 ?p27 KIP1? cells gradually declined under stimulation conditions and reached a minimum level after 72 h, but these decreases were not statistically significant.Moreover, the median frequencies of p27 KIP1 -positive cells in CLL patients remained markedly higher compared with healthy volunteers at each time point tested (Table 3).We next examined whether blocking CTLA-4 on the cell surface would affect the expression of the key regulators of G0/G1 phase.We observed a significant impact of CTLA-4 blockade on the expression of cell cycle regulators of G0/ G1 phase in CLL patients as well as in healthy individuals.As regards cyclin D2, in both studied groups, we observed a marked decrease in the median proportions of cyclin D2positive cells after 24 h of blocking culture (Figs. 6,7,8).Moreover, no significant differences in the median percentages of cyclin D2 ?cells between CLL patients and healthy individuals were found. As regards cyclin D3 expression, after 24 h of blocking culture, in CLL patients, we observed a marked decrease in the median proportions of cyclin D3-positive cells (Figs. 6,7).In healthy volunteers, CTLA-4 blockade led to a decrease in CD19 ?cyclin D3 ?cells after 24 and 72 h of culture (Figs. 6,8).Moreover, in CLL patients and in healthy individuals, after 72 h of CTLA-4 blocking culture, the median frequencies of cyclin D3-positive cells were markedly lower compared to corresponding cells after 24 h of culture (Figs. 6,7,8).Furthermore, the median frequencies of cyclin D3-positive leukaemic cells in CLL patients were markedly lower compared with healthy volunteers at each time point tested (P = 0.00002 and 0.005 after 24 and 72 h of blocking culture, respectively). As regards p27 KIP1 expression, in CLL patients, CTLA-4 blockade resulted in an increase in the median proportions of p27 KIP1 -positive cells compared to corresponding cells under stimulating conditions, although this increase was statistically significant only after 24 h of blocking culture (Figs. 6,7).Moreover, in CLL patients, after 72 h of CTLA-4 blocking culture, the median frequency of p27 KIP1? cells was markedly lower compared to corresponding cells after 24 h of culture (Figs. 6,7).In healthy individuals, an increase in the median proportions of p27 KIP1 -positive lymphocytes following 24 and 72 h of blocking culture was observed, although this increase was not statistically significant (Figs.6,8).Moreover, the median frequency of p27 KIP1 -positive cells in CLL patients remained markedly higher compared with healthy individuals at each time point tested (P = 0.000005 and 0.003 after 24 and 72 h of blocking culture, respectively). ",
"section_name": "Expression of CTLA-4 molecule and cell cycle regulators of G0/G1 phase in freshly drawn CLL cells and normal CD19 1 lymphocytes",
"section_num": null
},
{
"section_content": "In the present study, we confirmed our earlier observations [22] that both the surface and cytoplasmic expression of the CTLA-4 molecule is overexpressed in freshly isolated peripheral blood CLL cells.This finding raises a question about the mechanisms underlying the elevated levels of CTLA-4 expression in CLL cells.Since CTLA-4 expression is transiently inducible during T and B activation [27,28] and it is considered to be an activation marker, the higher expression of CTLA-4 in CLL cells may reflect the systemic activation status in the periphery in CLL patients.It has been well documented that CLL cells receive many growth-promoting signals [29][30][31], which are mediated by receptors constitutively expressed on the subset of neoplastic B cells [32,33].In addition, literature data confirm that CLL cells bear the phenotype of activated B cells based on overexpression of the classical activation markers CD23, CD25, CD69 and CD71, and under-expression of CD22, Fcc IIb, CD79b, and immunoglobulin D, which are down-regulated by cell triggering and activation [34].The suggestion that CLL cells may be in a partial state of activation also results from the observation of cyclin D2 expression, which is induced during cell stimulation by growth factors in early G0 phase.Accordingly, in the current study, we confirmed our previous observations [22,35,36] that the median proportion of freshly isolated CLL cells co-expressing cyclin D2 was markedly higher than that observed in normal B lymphocytes.Furthermore, based on the fact that CTLA-4 gene polymorphisms may influence the CTLA-4 expression level in CLL cells [22,37], we cannot exclude that the strong divergence of CTLA-4 expression observed among the patients might result, at least in part, from the presence of specific alleles predisposing to overexpression of the CTLA-4 molecule. We also found a negative correlation between CTLA-4 expression in CLL cells and the clinical parameters such as Rai stage and leucocyte as well as lymphocyte count.This finding is consistent with the study of Joshi et al. [38] showing that the level of CTLA-4 gene expression in CLL cells predicts clinical outcome of CLL patients; lower expression of CTLA-4 is associated with significantly shorter time to treatment and poor prognosis compared with high CTLA-4 expression associated with good clinical outcome. In the present study, we showed for the first time the impact of ex vivo specific stimulation of CLL cells with DSP30 (CpG ODN)?rIL-2 on CTLA-4 expression.An assessment of CTLA-4 expression under this stimulation condition seems to have clinical relevance in the context of the discussion about using CpG ODNs as therapeutic agents in CLL patients [39].We found that ex vivo stimulation led to a significant decrease in the median proportion of CLL cells expressing the CTLA-4 molecule on their surface, which became comparable to the value of the corresponding cells in healthy individuals.In contrast, the intracellular expression of this protein in leukaemic cells markedly increased after 72 h of cell culture in the presence of DSP30 and rIL-2 and remained significantly higher than in healthy volunteers.This observation seems to suggest that the down-regulation of sCTLA-4 expression upon stimulation may result from disturbed recycling of the CTLA-4 molecule to the cell surface in CLL.Moreover, as higher expression of the CTLA-4 gene is associated with good clinical outcome [38], application of CpG ODN that reduces the surface expression of CTLA-4 in leukaemic cells as a therapeutic agent may not be beneficial for CLL patients. Literature data show that ex vivo stimulation with CpG ODN results in down-regulation of p27 KIP1 expression and up-regulated expression of cyclins D2 and D3 in CLL cells and normal B lymphocytes [40][41][42][43].In the present study, we also observed the down-regulation of p27 KIP1 expression following ex vivo stimulation in CLL patients as well as in healthy volunteers.Of note, in spite of the decrease in p27 KIP1 expression in both studied groups, the median frequencies of p27 KIP1 -positive cells in CLL patients remained significantly higher compared with healthy individuals at each time point tested.It is noteworthy that high expression of this protein is associated with poorer outcome of CLL [44][45][46].As regards cyclin D2, in contrast to others [40][41][42], after 24 h of stimulating culture, we did not observe up-regulation of cyclin D2 expression in CLL cells.This discrepancy might result from the different experimental procedures used.We estimated the expression of these proteins as the proportions of CLL cells co-expressing several proteins using flow cytometry, whereas the authors of the contradictory reports examined the protein expression by western blot analysis.Due to cyclin D2 being overexpressed in freshly drawn CLL cells compared to normal B lymphocytes [22,35,36,47] and maintained at similar high levels in CLL cells after 24 h of ex vivo stimulation, the median percentage of cyclin D2-positive cells in CLL patients was comparable to the corresponding cells in healthy individuals, in which up-regulation of cyclin D2 expression was observed.Moreover, in agreement with literature data [40][41][42][43], we found a significant increase in the median percentages of cyclin D3-positive cells after 24 h of ex vivo stimulation in CLL patients as well as in healthy volunteers.Of note, the median proportions of CLL cells co-expressing cyclin D3 remained markedly lower at each time point tested compared to corresponding cells in healthy individuals.To elucidate this phenomenon, further studies are necessary. One of the main aims of this study was to examine whether the CTLA-4 molecule is able to influence the expression of the key regulators of G0/G1 phase in CLL cells as well as normal B lymphocytes.Our previous data [22] indicated that expression of the CTLA-4 molecule in CLL cells correlated with the expression of the G0/G1 phase regulators cyclins D2 and D3, and p27 KIP1 , suggesting the impact of CTLA-4 on prolonging the G1 phase in leukaemic lymphocytes.In the present study, we examined the expression of the mentioned cell cycle regulators following blockade of CTLA-4 on the surface of CLL cells as well as normal B lymphocytes.Blocking CTLA-4 on the surface of the studied cells is a useful method in research to elucidate the function of this protein.We found that CTLA-4 blockade did affect the expression of studied regulators of G0/G1 phase.In particular, we observed for the first time a significant decrease in the median proportions of both cyclin D2-and cyclin D3positive cells in CLL cells as well as in normal B lymphocytes following 24 h of blocking culture.Moreover, we noted that CTLA-4 blockade led to an increase in the median proportions of p27 KIP1 -positive cells after 24 h of culture in CLL cells as well as in normal B lymphocytes, although this increase was statistically significant only in the group of CLL patients.What are the possible mechanisms responsible for this phenomenon?It has been shown that the expression of D-type cyclins and p27 KIP1 protein stability is controlled by PI3 K (phosphatidylinositol 3-kinase)-related pathways [48,49].Activation of PI3 K-mediated pathways leads to transcription of D-type cyclins and to reduction of the p27 KIP1 protein level [48,49].In some experimental systems, it has been shown that CTLA-4 engagement on T cells activates PI3K [50].As CTLA-4 blockade has an opposite effect, it seems highly probable that CTLA-4 blockade may lead to inactivation of PI3K-related pathways.Based on this hypothesis, we can speculate that blockade of CTLA-4 on CLL cells as well as on normal B lymphocytes may lead to inactivation of PI3K-related pathways, resulting in decreased expression of cyclins D2 and D3, and accumulation of p27 KIP1 protein, in consequence.Further studies are required to confirm a direct relationship between CTLA-4, PI3K-related pathways, and the expression of the mentioned cell cycle regulators of G0/G1 phase. Furthermore, an interesting finding was that the median proportions of cyclin D2-positive cells in CLL patients and healthy volunteers were comparable following CTLA-4 blockade, whereas the median percentages of cyclin D3 ? and of p27 KIP1? leukaemic cells remained lower and higher, respectively, than in healthy individuals.Further investigations are needed to explain this phenomenon. Extending our previous study, we have demonstrated by blocking experiments that the CTLA-4 molecule influences the expression of the key regulators of G0/G1 phasecyclins D2 and D3, and p27 KIP1 -in both malignant and b Fig. 7 Representative examples of flow cytometric analysis of the effect of CTLA-4 blockade on expression of G0/G1 phase regulators in leukaemic cells after 24 h (a) and 72 h (b) of blocking culture. Numbers on dot plots represent the percentage of leukaemic cells expressing cyclin D2, cyclin D3 or p27 KIP1 normal B lymphocytes.We also confirmed the recent observation that CTLA-4 expression in peripheral blood CLL cells negatively correlates with disease progression, suggesting its clinical relevance as a prognostic marker.Since CTLA-4, cyclin D2 and p27 KIP1 seem to have prognostic significance in CLL, further studies are necessary to elucidate the molecular mechanisms of the relationship between these proteins. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "Acknowledgments This work was supported by a grant from the National Science Centre (Poland, Project Number: N N402 284936). ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/),which permits unrestricted use, distribution, and reproduction in any medium, provided 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. ",
"section_name": "Conflict of interest None.",
"section_num": null
}
] |
10.1186/s12864-016-2608-3
|
Screening of whole genome sequences identified high-impact variants for stallion fertility
|
Stallion fertility is an economically important trait due to the increase of artificial insemination in horses. The availability of whole genome sequence data facilitates identification of rare high-impact variants contributing to stallion fertility. The aim of our study was to genotype rare high-impact variants retrieved from next-generation sequencing (NGS)-data of 11 horses in order to unravel harmful genetic variants in large samples of stallions.Gene ontology (GO) terms and search results from public databases were used to obtain a comprehensive list of human und mice genes predicted to participate in the regulation of male reproduction. The corresponding equine orthologous genes were searched in whole genome sequence data of seven stallions and four mares and filtered for high-impact genetic variants using SnpEFF, SIFT and Polyphen 2 software. All genetic variants with the missing homozygous mutant genotype were genotyped on 337 fertile stallions of 19 breeds using KASP genotyping assays or PCR-RFLP. Mixed linear model analysis was employed for an association analysis with de-regressed estimated breeding values of the paternal component of the pregnancy rate per estrus (EBV-PAT).We screened next generation sequenced data of whole genomes from 11 horses for equine genetic variants in 1194 human and mice genes involved in male fertility and linked through common gene ontology (GO) with male reproductive processes. Variants were filtered for high-impact on protein structure and validated through SIFT and Polyphen 2. Only those genetic variants were followed up when the homozygote mutant genotype was missing in the detection sample comprising 11 horses. After this filtering process, 17 single nucleotide polymorphism (SNPs) were left. These SNPs were genotyped in 337 fertile stallions of 19 breeds using KASP genotyping assays or PCR-RFLP. An association analysis in 216 Hanoverian stallions revealed a significant association of the splice-site disruption variant g.37455302G>A in NOTCH1 with the de-regressed estimated breeding values of the paternal component of the pregnancy rate per estrus (EBV-PAT). For 9 high-impact variants within the genes CFTR, OVGP1, FBXO43, TSSK6, PKD1, FOXP1, TCP11, SPATA31E1 and NOTCH1 (g.37453246G>C) absence of the homozygous mutant genotype in the validation sample of all 337 fertile stallions was obvious. Therefore, these variants were considered as potentially deleterious factors for stallion fertility.In conclusion, this study revealed 17 genetic variants with a predicted high damaging effect on protein structure and missing homozygous mutant genotype. The g.37455302G>A NOTCH1 variant was identified as a significant stallion fertility locus in Hanoverian stallions and further 9 candidate fertility loci with missing homozygous mutant genotypes were validated in a panel including 19 horse breeds. To our knowledge this is the first study in horses using next generation sequencing data to uncover strong candidate factors for stallion fertility.
|
[
{
"section_content": "A major challenge in current equine genomics is to understand the genetic basis of mutations influencing stallion fertility.In Hanoverian stallions, several studies implicated loci that have been shown to play a significant role in stallion fertility.Mutations within CRISP3 [1,2], SPATA1, INHBA, ACE, SP17, FSHB, PRLR [3][4][5], PLCz1 [6] and FKBP6 [7] were significantly associated with stallion fertility.Abnormalities in number of Xand/or Y chromosomes have been shown as causes for abnormal sexual development [8][9][10][11][12].Until now, only few mutations were identified to be associated with sub-or infertility in stallions.FKBP6 genotyping was recommended for stallions affected by impaired acrosome reaction (IAR).In human, only 20 % of male factor infertility can be diagnosed using genetic testing [13].Whole-genome sequencing approaches facilitate a comprehensive identification of high-impact variants with potentially deleterious effects on traits analysed [14].Rare genetic variants are more likely to be functionally deleterious and to cause loss-of function of coding proteins [14,15].Recently, whole-genome sequencing was used to uncover the roles of rare genetic variants associated with spermatogenic failure in men [16].To date, no whole-genome sequence screens were performed for stallion fertility.We employed next-generation sequence (NGS) data to screen male fertility associated genes in order to unravel the role of genetic variants for which damaging effects on the protein structure were predicted.Herein, we employed bioinformatic analysis to identify human and mice genes linked through common gene ontology (GO) male reproductive processes.In order to focus the search for rare high-impact variants within the gene list, we filtered the whole genomes sequence data for variants which were only present as wild type homozygotes or heterozygotes in fertile horses.We assumed that in fertile stallions, variants with deleterious effects on fertility should be restricted to heterozygous genotypes for the recessive alleles or the homozygous wild type genotype for the dominant allele, and the presence of one of these variants may reduce stallion fertility.The objective of the present study was to genotype rare high-impact variants retrieved from NGS-data of 11 horses in order to unravel harmful genetic variants in a large sample of stallions. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Gene ontology (GO) terms for male reproductive processes and Ensembl and NCBI databases were used to search for genes with an effect on male fertility in human and mice.We retrieved a list of 1256 genes identities (Additional file 1).Using g:Profiler [17,18] we could convert 1194 genes to known equine orthologs and had to exclude 62 genes due to unknown equine annotation or unknown chromosomal position.Using PANTHER classification system [19,20], the 1194 male fertility related genes were assigned to 67 pathways, 85 molecular functions, 82 biological processes and 16 cellular components. ",
"section_name": "Search for genes involved in stallion reproduction",
"section_num": null
},
{
"section_content": "We searched the whole genome sequences of all 11 horses for genetic variants within the 1194 male reproduction related genes.The first filter sorted out all genetic variants for which both homozygous genotypes and the heterozygous genotypes were present.This filter left 1259 genetic variants with influence on the coding sequence and contained only variants for which the mutant homozygous genotype was not present.The second filtering for high impact variants retrieved a total of 19 SNPs within 18 genes and 38 indels within 31 genes (Additional file 2).Further evaluation was restricted to SNP variants only.Of the 19 SNPs, 16 SNPs were located in 15/1194 genes for stallion reproduction and three SNPs (ENSECAG00000020135, ENSECAG00000021286 and ENSECAG00000018118) were not obviously related to male reproduction, but located within uncharacterized genes or pseudogenes on the complementary DNA strand (Table 1, Additional file 3).The most frequently predicted high-impact effects were donor splice site disruptions (n = 7) and stop codon gains (n = 6) (Table 2).We could assign these 15 genes with possibly rare high-impact variants to four pathways, 14 molecular functions, 14 biological processes and one cellular component.Using GeneMANIA, we constructed a gene-gene functional network for 13/15 genes based on GO annotation (Additional file 4). ",
"section_name": "Mutation detection in 1194 equine genes",
"section_num": null
},
{
"section_content": "In total, we genotyped 19 SNPs whereof two SNPs, one in FNDC3A and one SNP on the complementary DNA strand (ENSECAG00000018118), proved monomorphic in a sample of 96 stallions.Out of the 17 remaining SNPs genotyped, 15 SNPs were related to male reproduction.Two SNPs (ENSECAG00000020135, ENSECAG00000021286) were located within uncharacterized genes/pseudogenes on the complementary DNA strand whereas these SNPs had no high impact within the candidate genes on protein structure.Genotyping for the 17 polymorphic SNPs was performed in 337 stallions from 19 different breeds using PCR-RFLP and competitive allele specific PCR (KASP) genotyping technique (Additional file 5).Three SNPs within NEURL1, GHRL and BTNL2 deviated from Hardy Weinberg equilibrium (HWE) (Table 3).For all SNPs, the minor allele was represented by the mutant allele with a mean minor allele frequency (MAF) of 0.07 (range = 0.002-0.306).For 6/17 variants all three genotypes were found, of which the mutant homozygous genotypes were underrepresented with a mean genotype frequency of 0.06 NEURL1 0/0 0/0 0/1 0/0 0/1 0/0 0/0 0/0 0/0 0/0 0/0 g.77472655G>C ECA3 KDR 0/0 0/1 0/0 0/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 g.74610774C>T ECA4 CFTR 0/0 0/0 0/0 0/0 0/0 0/0 0/1 0/0 0/0 0/0 0/0 g.56937215C>T ECA5 OVGP1 0/0 0/0 0/0 0/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 g.45985131A>G ECA9 FBXO43 0/0 0/0 0/0 0/0 0/1 0/0 0/0 0/0 0/0 0/0 0/0 g.82699661C>T ECA9 TSSK6 0/0 0/0 0/0 0/0 0/1 0/0 0/0 0/0 0/0 0/0 0/0 g.7083659A>T ECA11 SLC9A3R1 0/0 0/0 0/1 0/1 0/0 0/0 0/0 0/1 0/0 0/0 0/0 g.40694339G>A ECA13 PKD1 0/0 0/0 0/0 0/0 0/0 0/1 0/0 0/0 0/0 0/0 0/0 g.6704968C>T ECA16 GHRL 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/1 0/0 0/0 g.19034281C>T ECA16 FOXP1 0/0 0/0 0/0 0/0 0/1 0/0 0/0 0/0 0/0 0/0 0/0 g.21894180G>A ECA17 FNDC3A 0/1 0/0 0/0 0/0 0/1 0/0 0/0 0/1 0/0 0/0 0/0 g.32635273T>C ECA20 BTNL2 0/0 0/0 0/1 0/0 0/0 0/0 0/0 0/0 0/1 0/0 0/0 g.35255390T>C ECA20 TCP11 0/0 0/0 0/0 0/0 0/0 0/1 0/0 0/0 0/0 0/0 0/0 g.4323852G>A ECA25 SPATA31E1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/1 0/0 0/0 g.37453246G>C ECA25 NOTCH1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/1 0/0 g.37455302G>A ECA25 NOTCH1 0/0 0/1 0/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 g.79813487A>T ECA3 ENSECAG00000020135 0/0 0/0 0/0 0/0 0/0 0/1 0/0 0/0 0/0 0/0 0/0 g.25184403G>C ECA7 ENSECAG00000021286 0/0 0/0 0/0 0/0 0/0 0/1 0/0 0/0 0/0 0/0 0/0 g.30073089G>A ECA13 ENSECAG00000018118 0/0 0/0 0/0 0/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0 The variant ID (SNP ID), chromosomal position (ECA), gene symbol (Gene) and the horse genotypes are given.Genotypes either represent wild type variant (0/0), or mutant in heterozygote state (0/1), highlighted in bold.The animals marked with an asterisk were sequenced by Metzger et al. (2014) [52] the animals marked with two asterisks were sequenced by Orlando et al. (2013) [51].The reference horse genome (Twilight) is derived from the Thoroughbred mare Twilight (EquCab2.70) (range = 0.003-0.223).For 9/17 genetic variants, no stallion exhibited the mutant homozygous genotype (Table 4).The expected number of mutant homozygous genotypes for stallions, calculated as q 2 (mutant allele frequency squared) x number of stallions (n) stallions, revealed two variants within FOXP1 and OVGP1 with expected number of genotypes of 0.92 and 1.04 (Table 5).The variants within ENSE-CAG00000020135 and ENSECAG00000021286 were homozygous wild type in all stallions (Table 4).We grouped the genetic variants genotyped into four groups (I-IV) according to their minor allele frequency (MAF).In group I, the mutant allele did not occur (MAF = 0), in group II, the mutant allele occurred in a very low frequency, which were classified as rare variants (MAF < 0.01) [14].In group III, the mutant allele occurred in a low frequency (MAF > 0.01-0.1),and in group IV the mutant allele had a moderate frequency (MAF > 0.1).Variants in non-male reproduction related genes ENSE-CAG00000020135 and ENSECAG00000021286 were sorted into group I. Variants in CFTR, PKD1 and SPA-TA31E1 were classified as rare variants (group II).Eight variants were sorted into group III and four variants into group IV (Table 5).The distribution of the variants genotyped within discovery breeds and validation cohorts should allow distinguish between private SNPs and SNPs shared by several breeds.With the exception of two private Icelandic SNPs within ENSECAG00000020135 and ENSE-CAG00000021286, all high-impact SNPs were shared by 2-12 breeds (Additional file 6). ",
"section_name": "Validation of high-impact SNPs in fertile stallions",
"section_num": null
},
{
"section_content": "In 216 Hanoverian stallions, a nominally significant association with de-regressed EBV-PAT was found for the NOTCH1 variant g.37455302G>A (P = 0.00003) using a model fitting the genotypes.Accounting for multiple testing of 15 SNPs using Bonferroni correction, resulted in a significant association for the NOTCH1 variant g.37455302G>A (P = 0.00045) explaining 11 % of the variation of de-regressed EBV-PAT.A mixed model analysis accounting for genomic relationships among stallions gave a P-value of 0.00052 and after Bonferroni correction a P-value of 0.00828.Hanoverian stallions exhibiting the wild-type for the NOTCH1-associated SNP had a de-regressed EBV-PAT of 101 onto the scale 100 ± 20 indicating stallion fertility closely at the population average.The differences among the NOTCH1-homozygotes were 41 points of the de-regressed EBV-PAT (P-value < 0.0001).Stallions heterozygous for the mutant NOTCH1variant had a mean of 100 for the de-regressed EBV-PAT.Other variants failed the nominal significance thresholds (Additional file 7).Stallions heterozygous or homozygous for the KDR-variant showed a higher The variant ID (SNP ID) and dbSNP accession number, gene symbol and location in the gene, type of high-impact effect and corresponding substitution type on protein level are given.Variants causing a stop codon are marked with an asterix mean of de-regressed EBV-PAT than homozygous wild type stallion.Due to high standard errors and low genotype frequencies, no significant associations, but a tendency among the homozygous and heterozygous genotype means for de-regressed EBV-PAT were found for six SNPs within NEURL1, OVGP1, FOXP1, NOTCH1 (g.37453246G>C), SPATA31E1 and FBXO43 (Additional file 7). ",
"section_name": "Association analysis in Hanoverian stallions",
"section_num": null
},
{
"section_content": "Based on validation and association results, we distinguished three classes among the high-impact variants (Table 6). ",
"section_name": "Classification of high-impact variants",
"section_num": null
},
{
"section_content": "Screening of whole genome sequences for high-impact genetic variants within gene networks for male fertility in stallions appeared useful to identify variants that may highly influence stallion fertility.All high-impact variants we classified due to stop codon gains, start losses, donor or acceptor splice site disruptions can be classified as loss-of-function (LOF) mutations [14,21].A major effect on fertility by loss-of-function mutations was demonstrated in previous studies [22][23][24][25].Data from this Significant associations of the high-impact variants with the estimated breeding values of the paternal component for the pregnancy rate per estrus (EBV-PAT) in Hanoverian stallions (n = 216) are indicated by Bonferroni-corrected P-values.For each variant, a plus sign indicates the missing mutant homozygous genotype (GT missing ), high and low heterozygous genotype frequencies (2pq high, 2pq low ) and the expected number of mutant homozygous genotypes for stallions E (q 2 stallion ) are given.The number of breeds where the variants were observed is counted and a grouping (group I-IV) by the minor allele frequency (MAF) is given study indicate a high-impact variant as a stallion fertilityassociated locus (class I) in NOTCH1 and 9 high-impact variants as putative stallion fertility-associated loci (class II).Genes putatively associated with stallion fertility included CFTR, OVGP1, FBXO43, TSSK6, PKD1, FOXP1, TCP11, SPATA31E1 and NOTCH1 (g.37453246G>C).These genes are connected with each other via functional interacting networks based on GO male fertility processes. In mice, NOTCH1 gain-of-function resulted in reduced male fertility due to failure of spermatogenesis [26], while immotile spermatozoa and sterility was detected in NEURL1 null mice [27].The fertile stallions used for validation were frequently uses in artificial insemination, thus sperm motility and morphology are in the normal range in these stallions.Fertile stallions that exhibited unfavorable genotypes in NOTCH1-highimpact variants had de-regressed EBV-PAT as well EBV-PAT below the population mean suggesting reduced fertility due to other unknown damaging mechanism on sperm function.For the KDR high-impact variant g.77472655G>C in KDR, heterozygous or homozygous carriers showed putatively higher de-regressed EBV-PAT and EBV-PAT implicating a beneficial effect of this variant.The latter is supported by the 'less-is-more' hypothesis [28] and an advantageous effect of deleterious variants due to positive selection [29].An advantageous effect on reproduction of deleterious variants alleles due to positive selection was recently demonstrated in buffalo [30].NOTCH1, NEURL1 and KDR are key factors for male fertility, making their application for stallion fertility highly desirable.NOTCH1 is part of the Notch signaling network and regulates interactions between physically adjacent cells [31].NEURL1 is required for axonemal integrity in spermatozoa [27].It functionally belongs to spermatogenesis (GO:0007283) and is associated to spermatid differentiation (GO:0048515), sperm motility (GO:0030317) and spermatid differentiation (GO:0048515).KDR is present in spermatozoa [32] and was identified as a regulator of germ cell survival during establishment of spermatogenesis in cattle [33]. Based on our hypothesis of missing mutant genotypes in fertile stallions, we detected 9 putative loci for stallion fertility defined as class II variants.These non-private and high-impact variants were only present in very low to or low frequency.The expected frequency of mutant homozygous genotypes for stallions E(q 2 stallion ) indicated a low-probability of their occurrence in fertile stallions.As an explanation for the missing homozygous mutant genotypes in fertile stallions one can assume that homozygous mutant genotypes are limited to infertile stallions.This may be further tested in an unselected sample of male foals under the assumption that these mutations are still segregating in very few males with the homozygous mutant genotype may be infertile. Class II variants within CFTR and TSSK6 indicated high-impact on male fertility, confirmed by several studies in human and mice.In infertile men, high-effect variants at acceptor splice site in CFTR were found in nonobstructive azoospermia, oligospermia, asthenospermia and teratospermia men [34,35].Infertility due to spermatogenic impairment was reported in male TSSK6 knockout mice [36] and in Chinese men exhibiting a triallelic SNP in TSSK6 [37].PKD1 is required for male reproductive tract development.PKD1-null mice develop multiple organ defects in the male reproductive tract and infertility [38].TCP-11 modulates sperm fertilizing ability.It stimulates capacitation and inhibits spontaneous acrosome loss [39,40].Subfertility in human could result from alterations in the structure of TCP-11 [39]. Validation of class II high-impact variants within SPA-TA31E1, OVGP1, FOXP1, FBXO43 indicate further putative loci for stallion fertility, however the consequences g.37453246G>C NOTCH1 Three classes of high-impact variants with corresponding single nucleotide polymorphism (SNP) identities (ID) and gene are given of mutations on male infertility in human and mice are not yet known.Nevertheless, SPATA31E1 functionally belongs to spermatogenesis (GO:0007283).OVGP1 is linked to single fertilization (GO:0007338) via binding to oocyte zona pellucida [41].It enhances sperm viability, motility [42] and capacitation and shows beneficial effects on fertilization and early embryo development [43,44].FOXP1 is an androgen-responsive transcription factor that modulates androgen receptor signaling [45].FBXO43 place a role in sperm-induced meiotic exit [46] and adjustment of the number of round spermatids [47]. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Out of 1194 equine genes involved in male fertility processes we identified the g.37455302G>A NOTCH1 variant as a high-impact SNP with a significant effect on Hanoverian stallion fertility.As this detected highimpact-variant only occurs in a rare frequency in fertile stallions of many different breeds we recommend selection against this unfavourable genotype to improve stallion fertility.Next, we identified 9 high-impact SNPs in CFTR, OVGP1, FBXO43, TSSK6, PKD1, FOXP1, TCP11, SPATA31E1 and NOTCH1 (g.37453246G>C) absent in fertile stallions with putative deleterious effects in fertility of stallions.As a consequence, an early identification of carriers in unselected young stallions should be a beneficial to select only high-quality stallions for future breeding. ",
"section_name": "Conclusions",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "All animal work has been conducted according to the national and international guidelines for animal welfare.The Lower Saxony state veterinary office at the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Oldenburg, Germany, was the responsible Institutional Animal Care and Use Committee (IACUC) for this specific study.The EDTA-blood sampling for the present study had been approved by the IACUC of Lower Saxony, the state veterinary office Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Oldenburg, Germany (registration number 33.42502-05-07A482). ",
"section_name": "Ethics statement",
"section_num": null
},
{
"section_content": "Gene ontology (GO) terms related to male reproduction were used to obtain a comprehensive list of known human und mice genes predicted to participate in the regulation of male reproduction.We used the go.obodownload file which contains core GO ontology terms provided by the gene ontology consortium (http://purl.obolibrary.org/obo/go.obo, accessed 02.08.2014).In addition, a keyword search for annotated genes in the genome browsers Ensembl and NCBI for 'male reproduction', 'male infertility', 'male gonad development', 'spermatogenesis', 'acrosome reaction' , was conducted.We then searched NCBI PubMed with keywords for mutations within genes affecting male reproduction in human and livestock animals (including horses) from articles published before November 1st 2014.We employed Ensembl BioMart [48,49] (http://www.ensembl.org/biomart/martview/)to retrieve all human genes (Ensembl gene ids) associated with a GO term and converted human genes into equine orthologs using g.Profiler [17,18] [19,20] (http://www.pantherdb.org).The database Gene-Mania [50] (http://www.genemania.org/) was used to visualise co-expression and common pathways for networks of male fertility related genes. ",
"section_name": "Search for genes effecting stallion reproduction",
"section_num": null
},
{
"section_content": "We screened publicly available whole genome sequences of seven stallions including each one Icelandic, Standardbred and Przewalski stallion [51], two Hanoverian stallions and each one Arabian and Sorraia stallion [52], and four mares including each one Arabian, Norwegian fjord [51], Dülmen Horse [52], and the re-sequenced reference genome of the Thoroughbred mare Twilight [51] for mutations within selected candidate genes for stallion fertility. Variants within candidate genes were extracted according to the equine chromosomal positions obtained from the Ensembl annotation.We excluded all genetic variants with hits in the genome databases dbSNP (ftp://ftp.Functional analysis of variants obtained from resequencing data was performed using SnpEFF software [53].The SnpEFF impact category HIGH was used to filter SNPs for putative high-impact effects (large chromosome deletion, exon deletion, insertion/deletion frame shift, donor splice site disruptions, acceptor splice site disruptions, stop codon gains, stop codon losses, start losses).Validation was done using SIFT and Polyphen 2. High-impact effect variants were considered as potentially deleterious or expected to potentially correlate with complete loss of function (LoF) of the affected transcripts [14,54].In order to identify rare variants influencing stallion reproduction we filtered high-impact variants within candidate genes for stallion fertility with missing homozygous mutant genotypes in fertile stallions.Under this scenario, we expected that homozygosity for those variants is less likley in fertile stallions.Analyses were performed using SAS/Genetics, version 9.4 (Statistical Analysis System, Cary, NC, 2015). ",
"section_name": "Detection of deleterious mutations",
"section_num": null
},
{
"section_content": "In total, we analysed 337 fertile stallions of 19 breeds (Additional file 8).The largest proportion of the fertile horses were Hanoverian stallions (n = 226), used for artificial insemination at the National State Stud Celle of Lower Saxony in Celle.For 216 Hanoverian stallions breeding values of the paternal component of the pregnancy rate per estrus cycle (EBV-PAT) and de-regressed EBV-PAT were available.The mean reliabilities of the EBVs were at 0.7.For fertile stallions other than Hanoverian, at least 10 viable offspring were reported, targeting those stallions as fertile. ",
"section_name": "Validation population",
"section_num": null
},
{
"section_content": "We assumed that the mutant allele with high impact on protein structure should be completely absent or in a very low frequency in fertile stallions.We genotyped high-impact SNPs in fertile stallions using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and competitive allele specific PCR (KASP) genotyping technique [55] (LGC Genomics GmbH, Berlin, Germany) (Additional file 5).PCR-RFLP primers were designed using Primer3 [56,57] (http://bioinfo.ut.ee/primer3/).DNA was amplified according to standard protocols on PTC 100 thermal cyclers, MJ Research, Watertown, MA, US.For enzymatic digestion, we used 15 μl reaction volume containing 1.5 μl buffer, 1.5 U endonucleases and 5 μl PCR amplicons.The KASP genotyping reaction was run in 5 μl KASP Master mix 2x (FAM and HEX dye-labeled FRET cassette, Taq polymerase, buffer), 0.14 μl KASP Assay mix (KASP-by-design: two allele-specific primers, one common primer) and 5 μl template DNA.Allelic discrimination was carried out using the Applied Biosystems 7300 Real-Time PCR System (Life Technologies). ",
"section_name": "Validation of high-impact variants",
"section_num": null
},
{
"section_content": "The ALLELE procedure of SAS/Genetics was used to calculate allele and genotype frequencies, minor allele frequencies (MAF), observed heterozygosity (HET) and χ 2 -tests for Hardy-Weinberg equilibrium for the SNPs genotyped. Calculation of EBV-PAT in Hanoverians and the model used for estimation are described elsewhere [58].In brief, data and model employed are given in the following.Fertility data included the breeding seasons 1997 to 2005 of the National State stud Celle of Lower Saxony with a total of 19,897 broadmares, 246 stallions, 199,000 artificial insemination records and 96,114 estrus cycles.Trait analysed was the pregnancy rate per estrus.The trait values were encoded 1 when the artificial insemination (AI) was successful in an estrus cycle otherwise the trait value was 0. The success rate of AIs was verified through the reports of the breeders on the pregnancy of the mare and/or a foaling and/or an abortion.All estrus periods with consecutive AIs in the same breeding season were treated as unsuccessful.In addition, estrus periods reported by the breeder as unsuccessful (missing pregnancy of the mare) were also encoded with a trait value 0. We predicted breeding values (EBVs) for the paternal and embryonic component for each stallion.The animal threshold model for prediction of EBVs included the fixed environmental effects of insemination centre, age of stallion, breeding season, period within breeding season, the number of coverings within breeding season (covering number), time interval between coverings within a estrus (insemination regime), breeding history of mares (previous breeding achievement of mares), the random permanent environmental effects of the mare and stallion and the random additive genetic effects of the stallion and the embryo.The analyses were performed using MTGSAM and Gibbs sampling [59,60] for estimation of random effects and variance components.Heritability for the paternal component of the pregnancy rate per estrus was 1.1 %.All EBVs were standardized onto a mean of 100 and a standard deviation of 20.The random additive genetic effect of the stallion was defined as the paternal component of the pregnancy rate per estrus (EBV-PAT).EBVs >100 mean higher conception rates of the stallions than the population average.An association analysis for each SNP genotype was performed using the procedure GLM of SAS, version 9.4 (Statistical Analysis System, SAS Institute, Cary, NC, USA).The proportion of variance explained for (deregressed) EBV-PAT was estimated using the GLM procedure of SAS.In order to account for the population structure a mixed linear model (MLM) was employed using the respective genotype as fixed effect and a random animal effect through an identity-by-state-kinship (IBS) matrix.The IBS matrix reflects the genomic relationship matrix among all individuals genotyped and captures the relatedness among animals as well as the cryptic family structure.We used genotypings from the Equine SNP50 Beadchip (Illumina, San Diego, CA, USA) including 54,602 SNPs.Quality criteria were minor allele frequency (MAF) >0.05, genotyping rate per SNP and animal >0.90 and HWE (P < 0.00001).After filtering for quality criteria, 46,074 SNPs remained for building the IBS-matrix [6].The analysis was run using TASSEL, version 3.0.146[61].The Bonferroni correction was calculated using the MULTIPLE TEST procedure of SAS, version 9.4 to determine the threshold for experiment-wide significance. ",
"section_name": "Statistical analysis",
"section_num": null
}
] |
[
{
"section_content": "This study was supported by the Mehl-Mülhens Stiftung, Köln (DI-MM/2-1).We acknowledge the support of the National State Stud Celle of Lower Saxony, Celle, the Hanoverian breeding association for providing data and samples of Hanoverian warmblood stallions.We thank Heike Klippert Hasberg, Mogens Kilian Drabert, Stefan Neander and Jörn Wrede for expert technical support. ",
"section_name": "Acknowledgments",
"section_num": null
},
{
"section_content": "Newly discovered polymorphisms were submitted to NCBI dbSNP database (ss1457622626 -ss1457622648).The whole genome sequences of four stallions, one Arabian, two Hanoverian and one Sorraia are available at the NCBI Sequence Read Archive (SRA) (http://www.ncbi.nlm.nih.gov/sra),accession number SRP033361.Corresponding VCF files can be downloaded at Intrepid Bioinformatics at http://dx.doi.org/10.13013/J6MW2F2B.The whole genome sequences of three stallions, one Icelandic, Standardbred and Przewalski and the re-sequenced reference genome of the Thoroughbred mare 'Twilight' can be found at SRA under accession number SRA082086.Corresponding BAM and VCF files can be downloaded at http://geogenetics.ku.dk/publications/middle-pleistocene-omics. ",
"section_name": "Accession numbers",
"section_num": null
},
{
"section_content": "Additional file 1: Gene list of human (gene ID human) and corresponding equine gene identities (mapped gene ID horse) involved in male reproduction processes.Start and end of equine genes in base pairs (bp), chromosomal position of the equine gene (ECA), gene symbol, gene name, gene ontology (GO) term, GO molecular function, GO biological process, GO cellular component and GO pathway are given.(XLSX 5632 kb) Additional file 2: Whole genome sequences of horses filtered for high-impact variants in 11 horses.Type of mutation, single nucleotide polymorphism (SNPs) and Insertions/Deletions (Indels) and type of high-impact effect are given.(XLSX 20 kb) Additional file 3: Equine gene models for 14 genes related with stallion fertility and high-impact variants.Gene models were built based on the Ensembl annotation. The authors declare that they have no competing interests. Authors' contributions RS, JM, HS and OD conceived and designed the study.RS, MG, JM and OD performed the experiments.OD, RS and MG analysed the data.GM, HS, JM and OD contributed samples, insemination data and analysis tools.RS and OD drafted and finalized the manuscript.All authors read and approved the final manuscript. ",
"section_name": "Additional files",
"section_num": null
},
{
"section_content": "Additional file 1: Gene list of human (gene ID human) and corresponding equine gene identities (mapped gene ID horse) involved in male reproduction processes.Start and end of equine genes in base pairs (bp), chromosomal position of the equine gene (ECA), gene symbol, gene name, gene ontology (GO) term, GO molecular function, GO biological process, GO cellular component and GO pathway are given.(XLSX 5632 kb) Additional file 2: Whole genome sequences of horses filtered for high-impact variants in 11 horses.Type of mutation, single nucleotide polymorphism (SNPs) and Insertions/Deletions (Indels) and type of high-impact effect are given.(XLSX 20 kb) Additional file 3: Equine gene models for 14 genes related with stallion fertility and high-impact variants.Gene models were built based on the Ensembl annotation. ",
"section_name": "Additional files",
"section_num": null
},
{
"section_content": "The authors declare that they have no competing interests. Authors' contributions RS, JM, HS and OD conceived and designed the study.RS, MG, JM and OD performed the experiments.OD, RS and MG analysed the data.GM, HS, JM and OD contributed samples, insemination data and analysis tools.RS and OD drafted and finalized the manuscript.All authors read and approved the final manuscript. ",
"section_name": "Competing interests",
"section_num": null
}
] |
10.5114/aoms.2015.53289
|
Systematic review/Meta-analysis The role of gender in patients with diffuse large B cell lymphoma treated with rituximab-containing regimens: a meta-analysis
|
Diffuse large B cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL). Although gender has not been included in prognostic systems, male gender has been found as a bad prognostic indicator in Hodgkin lymphoma, follicular lymphoma and chronic lymphocytic leukemia. The relationship between gender and prognosis is not clear in patients with DLBCL treated with rituximab-containing regimens. The aim of this meta-analysis is to determine the prognostic/predictive role of gender in patients with DLBCL treated with rituximab-containing regimens.We systematically searched for studies investigating the relationships between gender and prognosis in DLBCL treated with rituximab-containing regimens. After careful review, survival data were extracted from eligible studies. A meta-analysis was performed to generate combined hazard ratios for overall survival, disease-free survival (DFS) and event-free survival (EFS).A total of 5635 patients from 20 studies were included in the analysis. Our results showed that male gender was associated with poor prognosis in terms of overall survival (OS) (hazard ratio (HR) = 1.155; 95% confidence interval (CI): 1.037-1.286; p < 0.009). The pooled hazard ratio for DFS and EFS showed that male gender was not statistically significant (HR = 1.219; 95% CI: 0.782-1.899; p = 0.382, HR = 0.809; 95% CI: 0.577-1.133; p = 0.217).The present meta-analysis indicated male gender to be associated with a poor prognosis in patients with DLBCL treated with rituximab-containing regimens.
|
[
{
"section_content": "age, performance score, stage, proliferation fraction and gene expression profiles [3,4].Today, the International Prognostic Index (IPI) and age-adjusted International Prognostic Index (aaIPI) are the most important scores used in daily practice to determine the prognosis and treatment strategies.The IPI scoring system includes age, performance status, serum lactate dehydrogenase (LDH) level, stage and extranodal involvement; the aaIPI includes stage, LDH, performance status and age older than 60 years. In the last decade, the standard of care in patients with DLBCL has been the addition of anti-CD20 antibody-rituximab to classic cytotoxic chemotherapy.More sophisticated methods and drugs targeting oncogenic pathways and gene expression profiles have been used to predict the prognosis and response to therapy in recent years [5][6][7].Although gender has not been included in prognostic systems, male gender has been found to be a bad prognostic indicator in Hodgkin lymphoma, follicular lymphoma and chronic lymphocytic leukemia [8][9][10].However, the prognostic significance of gender has not been shown in all studies [11,12]. The aim of this meta-analysis was to determine the prognostic/predictive role of gender in patients with DLBCL treated with rituximab-containing regimens. ",
"section_name": "Introduction",
"section_num": null
},
{
"section_content": "",
"section_name": "Material and methods",
"section_num": null
},
{
"section_content": "A computer-based literature search using the PubMed/Medline database was performed by two independent researchers (VK, OD).The initial PubMed search using the combined term (rituximab) and (lymphoma, large B-cell, diffuse) resulted in 1520 returns up to August 7, 2013.Only English language and human studies were included in this analysis.Full text articles of all selected studies were retrieved.If a paper was selected for inclusion, the bibliographic references were carefully investigated to look for additional studies. ",
"section_name": "Research strategy",
"section_num": null
},
{
"section_content": "Prospective and retrospective randomized controlled studies involved patients older than 18 years who were treated according to rituximab-containing regimens.Case reports and series, letters, commentaries, lymphoma series including those other than DLBCLs and studies not containing an effect size for survival according to gender were not included. ",
"section_name": "Inclusion and exclusion criteria",
"section_num": null
},
{
"section_content": "Two independent reviewers decided which studies to include (MY, SP).The abstracts of all papers found to be appropriate for meta-analysis were read.The full texts of the candidate papers for this analysis were evaluated.Patients with DLBCL treated with rituximab-containing regimens were included in this analysis.If the patients had been included in different studies by the same author, the higher quality study was considered for this meta-analysis. ",
"section_name": "Selection of studies",
"section_num": null
},
{
"section_content": "The Newcastle-Ottawa Quality Assessment Scale was used for the evaluation of non-randomized controlled studies and the Jadad scoring system was used for the evaluation of randomized controlled studies by two independent reviewers (MY, VK).The Newcastle-Ottawa Quality Assessment Scale is used to determine the choice of patient population, comparability, follow-up and results of the studies.For these criteria studies are scored with stars between 0 and 9.A score of nine stars indicates the highest quality [13].The Jadad scoring system is based on 5 stars [14].Discrepancies between the authors after evaluations were re-evaluated and consensus was reached for all data. ",
"section_name": "Assessment of study quality",
"section_num": null
},
{
"section_content": "For studies evaluating gender and rituximabcontaining regimens in cases of NHL treated with rituximab-containing regimens, the following variables were extracted: essential data about study, author, publication year, country of study, design of the study, demographic data, gender distribution, treatment schedules, stage, and effect size of gender on the overall survival (OS), disease-free survival (DFS) and event-free survival (EFS). ",
"section_name": "Data extraction",
"section_num": null
},
{
"section_content": "The primary aim of this study was to analyze the effect of gender on the OS in patients with DL-BCL treated with rituximab-containing regimens.Disease-free survival and EFS were also analyzed.Hazard ratio (HR) was calculated with a 95% confidence interval (CI).Hr > 1 and not including 95% coincidence interval 1 were considered as significant.If there was no HR, summary statistics were used.Homogeneity was evaluated using the χ 2 -based test of homogeneity and inconsistency index.A p-value < 0.10 for χ 2 or I 2 > 50% was accepted as heterogeneity.Results were given using a fixed model.A p-value for summary HR of less than 0.05 was considered statistically significant.Publication bias was examined using Egger's regression intercept, Begg-Mazumdar rank correlation analysis, and a visual inspection of a funnel plot [15,16].Statistical analyses were performed using Comprehensive Metaanalysis V 2.0 (Biostat, Englewood, NJ). ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "The computer-based literature search using PubMed/Medline resulted in 1520 articles.Evaluation of the title and abstract of these articles revealed 377 case reports, 188 reviews, 32 letters, 34 comments, 63 non-English language studies, 13 pediatric studies, 11 cell studies, and 3 animal studies, and these were excluded from further analysis, leaving 799 papers.Of these 11 were cases series, 69 had included other lymphoma subtypes, 120 papers had not included rituximab-containing regimens, 3 did not report subgroup survival analyses, and 564 did not include the data effect size for gender.All these papers were excluded, leaving 26 papers.However, among these 26 papers, there were no effect size data in cases receiving rituximab according to gender in 5 papers and these papers were thus excluded from further analysis.Figure 1 shows the flowchart of articles included in this meta-analysis.Ultimately, 20 studies were included in this meta-analysis (Table I [11,[17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35]). ",
"section_name": "Study eligibility",
"section_num": null
},
{
"section_content": "Two of 20 studies included in this analysis were prospective and 18 were retrospective.The quality of retrospective studies was evaluated using the Newcastle-Ottawa Scale.In this scale 1-3 is considered as a low quality study, 4-6 as an intermediate quality study and 7-9 as a high quality study.The median Jadad score was 6 and 5 for retrospective and prospective studies, respectively. ",
"section_name": "Quality of papers included in this metaanalysis",
"section_num": null
},
{
"section_content": "The total number of patients included in this meta-analysis was 5635: 2879 (54.4%) men and Overall survival, disease-free survival, event-free survival Pooled HR for OS was evaluated in 16 studies and male gender was found to be associated with OS (HR = 1.155; 95% CI: 1.037-1.286;p < 0.009) (Figure 2).The association between gender and progression-free survival (PFS) was evaluated in eight studies and there was no statistically significant association (HR = 0.849; 95% CI: 0.671-1.074;p = 0.171) (Figure 3).Event-free survival was evaluated in two studies and there was no association between gender and EFS (HR = 0.809; 95% CI: 0.577-1.133;p = 0.217) (Figure 4). ",
"section_name": "Patients",
"section_num": null
},
{
"section_content": "There was no publication bias for OS (Begg's test, p = 0.564; Egger test, p = 0.557).The funnel plot did not show publication bias for OS (Figure 5 A).There was no publication bias for PFS (Begg's test, p = 0.286; Egger test, p = 0.254).The funnel plot was symmetric and did not show publication bias for OS (Figure 5 B).Publication bias could not be determined for EFS because only two studies were evaluated. ",
"section_name": "Publication bias",
"section_num": null
},
{
"section_content": "Male gender has been found to be a bad prognostic indicator for OS in cases treated with rituximab-containing regimens in some studies [17,18].An OS advantage has been shown in patients with DLBCL treated with rituximab-containing regimens for every age and risk group [36,37]. Immunochemotherapy is the standard of care in patients with DLBCL; however, variable responses have been documented.This means that more sensitive prognostic and/or predictive factors are required.Although it has not been evaluated in all studies, male gender has been found to be a prognostic factor at least in some studies [38].The relationship between gender and prognosis has been considered in recent years.Although this analysis covers studies between 2002 and 2013, the majority of the studies in this meta-analysis were published in 2013.Male sex has been shown to be a bad prognostic indicator in patients with DLBCL treated with rituximab-containing regimens [11,27]. Prognostic factors in 700 cases from eight centers in Asian and Western countries treated by R-CHOP were compared by Castillo et al.They found no difference for PFS and OS, but male sex was found to be a bad prognostic indicator in multivariate analysis of patients of Asian origin [24].This point is important, and more studies from different countries will be required to evaluate racial differences. The prognostic role of gender in DLBCL has been reported in only a limited number of papers.In one of these papers, Carella et gender was found to be a bad prognostic indicator and the HR was 1.52.A higher dose of rituximab in males was suggested by the authors [27].Gender was also found to be an independent prognostic factor by Gisselbrecht et al. in relapsed/refractory DLBCL treated with rituximab maintenance after autologous stem cell transplantation [39]. Lower serum rituximab levels, shorter exposure times and worse outcome in men were observed in the RICOVER-60, RICOVERnoRTh and pegfilgrastim trials (R1, R2, R3), and the SEXIE-R-CHOP 14 trial has been planned and performed.In the last ASCO conference this study was presented and a significantly improved outcome in male patients was observed with higher rituximab doses (500 mg/m 2 instead of 375 mg/m 2 ) [40].These results confirm the worse outcome with standard R-CHOP in men. The mechanism underlying the different prognosis in men and women is not clear.Pharmacokinetic studies suggest that higher serum levels are associated with lower drug clearance in females [41].Additionally, gender-associated gene polymorphisms may be a contributing factor in the higher response to immune-chemotherapy in females.GSTT1 deletion has been suggested as a causative factor for resistance to R-CHOP and also poor prognosis in male Korean patients [42]. In conclusion, OS was found to be better in female cases than males with DLBCL treated with immunochemotherapy in this meta-analysis.Higher doses of rituximab could be more useful in males; the ongoing SEXI-R-CHOP-14 study will help clarify this point. ",
"section_name": "Discussion",
"section_num": null
}
] |
[
{
"section_content": "The authors declare no conflict of interest. ",
"section_name": "Conflict of interest",
"section_num": null
},
{
"section_content": "",
"section_name": "R e f e r e n c e s",
"section_num": null
}
] |
10.3389/fonc.2020.01272
|
Low Humoral Immune Response and Ineffective Clearance of SARS-Cov-2 in a COVID-19 Patient With CLL During a 69-Day Follow-Up
|
Background: A recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), which began in Wuhan, China, with a high level of human-to-human transmission has been reported. There are limited data available on Coronavirus Disease 2019 (COVID-19) patients with hematological malignancies with more than 60 days of follow-up. This study describes the clinical characteristics, including multiple recurrences of COVID-19, in a patient with chronic lymphocytic leukemia (CLL) during 69 days of follow-up. Case Presentation: A 72-year-old female was admitted to hospital isolation after being infected with COVID-19 as part of a family cluster on January 30, 2020. Apart from SARS-Cov-2 virus infection, laboratory results revealed lymphocytosis of uncertain etiology and abnormal distribution of T lymphocytes. On blood smears, small blue lymphocytes with scant cytoplasm were observed, and the presence of high levels of circulating clonal B cells was also demonstrated by flow cytometry. The patient was diagnosed with COVID-19 and CLL. Among her family members, she had the highest viral loads and the fastest progression on lung injury and developed severe pneumonia. Serological results showed she had both 2019-nCoV-specific IgM and IgG antibodies; however, only IgG antibodies were detected in her husband's plasma. Results: A combination regimen of antiviral therapy and high-dose intravenous immunoglobulin (IVIG) in the early stage seemed to be effective for treating CLL and SARS-Cov-2 infection. Because of the low humoral immune response, the CLL patient could not effectively clear the SARS-Cov-2 infection and suffered from recurrence twice during the 69-day follow-up. Conclusion: In CLL, a neoplastic antigen-specific B-cell clone proliferates, and the progeny cells accumulate and outgrow other B cells, leading to immune deficiency. Considering the low humoral immune response and ineffective clearance of SARS-Cov-2 in CLL patients, the follow-up and home quarantine period should be extended. We need further studies to clarify suspending or continuing CLL therapy during COVID infection. For those patients who are prone to progression to severe disease, administering humoral immunity therapies can help to prevent disease progression and quickly meet the cure criteria.
|
[
{
"section_content": "Background: A recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), which began in Wuhan, China, with a high level of human-to-human transmission has been reported.There are limited data available on Coronavirus Disease 2019 (COVID- 19) patients with hematological malignancies with more than 60 days of follow-up.This study describes the clinical characteristics, including multiple recurrences of COVID-19, in a patient with chronic lymphocytic leukemia (CLL) during 69 days of follow-up. Case Presentation: A 72-year-old female was admitted to hospital isolation after being infected with COVID-19 as part of a family cluster on January 30, 2020.Apart from SARS-Cov-2 virus infection, laboratory results revealed lymphocytosis of uncertain etiology and abnormal distribution of T lymphocytes.On blood smears, small blue lymphocytes with scant cytoplasm were observed, and the presence of high levels of circulating clonal B cells was also demonstrated by flow cytometry.The patient was diagnosed with COVID-19 and CLL.Among her family members, she had the highest viral loads and the fastest progression on lung injury and developed severe pneumonia.Serological results showed she had both 2019-nCoV-specific IgM and IgG antibodies; however, only IgG antibodies were detected in her husband's plasma. Results: A combination regimen of antiviral therapy and high-dose intravenous immunoglobulin (IVIG) in the early stage seemed to be effective for treating CLL and SARS-Cov-2 infection.Because of the low humoral immune response, the CLL patient could not effectively clear the SARS-Cov-2 infection and suffered from recurrence twice during the 69-day follow-up. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "In CLL, a neoplastic antigen-specific B-cell clone proliferates, and the progeny cells accumulate and outgrow other B cells, leading to immune deficiency.Considering the low humoral immune response and ineffective clearance of SARS-Cov-2 in CLL patients, the follow-up and home quarantine period should be extended.We need further studies to clarify suspending or continuing CLL therapy during COVID infection.For those patients who are prone to progression to severe disease, administering humoral immunity therapies can help to prevent disease progression and quickly meet the cure criteria. ",
"section_name": "Conclusion:",
"section_num": null
},
{
"section_content": "Chronic lymphocytic leukemia (CLL) is a malignant hematological disorder characterized by the accumulation of single-clone B lymphocytes in peripheral blood, lymph nodes, and bone marrow (1).The diagnosis of CLL is based on the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) criteria (2).Infectious complications due to immune dysfunction constitute the leading cause of mortality in patients with CLL.A neoplastic antigen-specific B-cell clone proliferates and outgrows other B cells, leading to humoral immune deficiency in CLL patients (3).Moreover, monoclonal cells affect the phenotype and function of a variety of innate and adaptive immune cells, including monocytes, T cells, and natural killer cells, leading to a tumor-supportive environment and reduced immune surveillance (4,5).A study found that cellular immunotherapy impeded CLL, which may be related to acquired immune dysfunction that mainly manifests as abnormal expansion of T cells, failure to form synaptic T cells, and inhibition of T cell migration (6).Inhibition of immune surveillance increases the risk of viral infections (7). During the recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2), which began in Wuhan, China, a high degree of human-to-human transmission via direct or indirect contact with large respiratory droplets and/or airborne transmission has been reported (8)(9)(10).In addition to fever, a variety of signs and symptoms, including cough, diarrhea, fatigue, and headache, may be presented, and infections can cause disease that ranges in severity from trivial colds and sore throats to serious laryngeal and tracheobronchial infections, bronchiolitis, and frank pneumonia (11).Studies have shown the general susceptibility of the population and have revealed the risk factors for the development of severe pneumonia, including neoplastic disorders, cardiovascular and cerebrovascular diseases, and advanced age (10,12).There are limited data available on patients with coronavirus disease 2019 (COVID-19) and also hematological malignancies over more than 60 days of follow-up.In this study, we report the clinical characteristics, treatment, and multiple recurrence of COVID-19 in a patient with CLL during a 69-day follow-up. ",
"section_name": "BACKGROUND",
"section_num": null
},
{
"section_content": "A 72-year-old female experienced the onset of COVID-19 after being infected as part of a household cluster.Figure 1A shows the genealogical family tree.II-3 was the index patient, and he was admitted to the hospital on January 30, 2020, with a consistent cough that had lasted for 10 days.A nasopharyngeal swab was positive for SARS-Cov-2.Nasopharyngeal swabs from three males (I-1, II-3, and III-5) and 1 female (I-2) in the household were also positive for SARS-Cov-2.On admission, I-2 had no complaints, with no cough, fever, or shortness of breath.Four months prior, a blood analysis at a local hospital showed that she had increased lymphocytes (34.22 × 10 9 /L, range, 1.1-3.2) and normal counts of hemoglobin and platelets during a regular medical examination.On January 23, she had a moderate fever with a mild cough and no muscle aches or other symptoms.A chest computed tomography scan (CT) (Figure 1Ba1,a2) revealed slight bronchopneumonia in the superior lobe of the right lung.On January 30, a nasopharyngeal swab tested positive for SARS-Cov-2.Laboratory examinations showed abnormally increased lymphocytes (26.9 × 10 9 /L, range, 1.1-3.2) with normal counts of hemoglobin and platelets, elevated D-dimer (3.23 mg/L, range,<0.5),and decreased immunoglobulin A (0.75 U/L; range,0.82-4.53).A blood smear showed a large number of mature lymphocytes (Figure 1C).Immunophenotypic analysis showed that a single B lymphocyte clone accounted for 73.3% in non-erythroid cells; these cells were CD19 dim , CD5+, CD22+, CD20+, CD23+, CD200+, and FMC7+, while kappa light chain was restrictively expressed, and CD10, CD103, and sIgM were not expressed (Figure 1D).Multiple areas of ground-glass opacities under the pleura of both lungs were observed on chest CT on the admission day (Figure 1Bb1-b3).She was diagnosed with COVID-19 and CLL. Figure 1E shows the symptoms, the results of the virus RT-PCR tests, and the treatments used for I-2, I-1, and II-3 during a follow-up period of 69 days. On admission day 3, I-2 complained of shortness of breath, and arterial blood gas analysis revealed hypoxemia.A comprehensive analysis of the clinical manifestations and pulmonary image and a decreased neutrophil-tolymphocyte ratio showed that the patient had developed severe pneumonia.She received a modified combination regimen composed of 800/200 mg/day lopinavir/ritonavir (LPV/r), 1,000 WU/day interferon α-2b via aerosol inhalation, 1.6 mg/day subcutaneous thymosin, 0.6 g/day arbidol (tablets), and 250 mg/kg/day intravenous immunoglobulin (IVIG) for 5 days.Chest CT revealed focal consolidation accompanied by fibrosis in the patient on day 8 (Figure 1Bc1,c2).The patient suffered secondary pulmonary bacterial infection and received anti-infective treatment on day 10.Beginning on day 13, no fever, headache, dizziness, nausea, vomiting, or other complications affecting the respiratory system were observed.Re-examination with chest CT on day 18 revealed the absorption of pneumonia (Figure 1Bd1,d2).Respiratory, serum, and stool specimens were negative for SARS-Cov-2 for 2 days according to the results of repeated Chest CT revealed obvious improvement of the double lung infection in the patient on day 26 (Figure 1Be1,e2).However, on day 35, a respiratory specimen was positive for SARS-Cov-2, and the patient was readmitted to the hospital for isolation, though she had no complaints.Chest CT revealed new groundglass shadows in the patient (Figure 1Bf1,f2).Serological results revealed both 2019-nCoV-specific IgM and IgG antibodies in patient I-2; however, only specific IgG antibodies were detected in her husband's plasma.She received 1,000 WU/day interferon α-2b, 0.6 g/day arbidol (tablets), and 250 mg/kg/day IVIG for 3 days to modulate the immune response.Chest CT showed obvious absorption of the infection in the patient on day 47 (Figure 1Bg1,g2).On day 50, respiratory, stool, and serum specimens showed two successive negative results.The patient was discharged on day 52.IVIG was intravenously injected once a week after discharge.However, the patient tested positive for the virus for the third time on day 64, and she was the third admitted to the hospital for isolation.Interferon α-2b via aerosol inhalation, arbidol (tablets), and IVIG were applied on April 2. On day 67, respiratory, serum, and stool specimens were all negative for SARS-Cov-2 for 2 days once again.She was discharged again on day 69. Compared with that of I-1 and II-3, the neutrophil-tolymphocyte ratio (NLR) of I-2 was significantly lower (P < 0.05) (Figure 2A, t-test, using SPSS software version 22).According to the Ct values determined with the SARS-Cov-2 virus test, I-2 had the lowest Ct value among the three patients regardless of whether the specimen was from the initial nasopharyngeal swab or initial sputum (Figure 2B).Decreased Ct values indicate increased viral load.At diagnosis, the levels of immunoglobulin (Ig) G, IgA, and IgM for I-2 were among the lowest of those of the family members (Figure 2C).Routine blood examination was performed, and C-reaction protein, D-dimer, liver function, and renal function were monitored.The white blood cell (WBC) and lymphocyte counts for I-2 were evidently high, and they dropped significantly after every recurrent novel coronavirus infection (Figure 2D).The expressions of CRP and D-dimer were significantly elevated during the process of treatment ( Figures 2E,F).However, no remarkable changes were observed in hepatorenal function in I-1, II-3, and I-2 (Figures 2G,H).The dynamic trends of CD4+ and CD8+ lymphocytes, B lymphocytes, and cytokines IL-6 and IL-10 in patients I-2, I-1, The lymphocytes were detected with flow cytometry assays, and the cytokines were measured in serum.At first, the numbers of CD4+ and CD8+ lymphocytes of I-2 were higher than those of II-3 and I-1, while the CD4+/CD8+ ratio of I-2 was lower than that of the other two subjects (Figures 3C,D).Significant increases in IL-6 levels were observed in I-2 before the first recurrence (Figure 3E), and the IL-10 levels in I-2 were high before the second recurrence (Figure 3F). ",
"section_name": "CASE PRESENTATION",
"section_num": null
},
{
"section_content": "There are limited data available on COVID-19 patients with hematological malignancies over more than 60 days of followup.Our patient met the diagnostic IWCLL criteria.Monoclonal cells in CLL affect the phenotype and function of a variety of innate and adaptive immune cells, leading to an infectionprone environment and reduced immune surveillance (4,13,14).Studies have found humoral immune failure in CLL, which may be associated with hypogammaglobulinemia and IgG subclass deficiency (3,(15)(16)(17), as CLL cells impair the immune system (18).Several lines of evidence have indirectly indicated that antigenic stimulation through the B-cell receptor (BCR) supports CLL development (19), and patients with CLL should avoid using drugs that impair B-cell function during SARS-CoV-2 infection. Furthermore, there is increased susceptibility to viral infections due to T-cell dysfunction in CLL patients (17).By monitoring and comparing the patient's laboratory findings with those of the other family members, we found significant differences between the immunosuppressed patient and immune-competent patients, including the counts of blood cells and of the CD4+ and CD8+ lymphocyte subpopulations and B cells in peripheral blood and the levels of cytokines after 2 months of infection.Within 10 days after infection, the WBC of I-2 decreased significantly with decreases in neutrophils and lymphocytes to a certain extent, while I-1 and II-3 had normal immune function, increased neutrophils, and a slight decrease in lymphocytes.At first, the numbers of CD4+ and CD8+ lymphocytes of I-2 were higher than those of II-3 and I-1, while the CD4+/CD8+ ratio of I-2 was lower than that of the latter two subjects.During the follow-up, a significant decrease in CD8+ lymphocytes was observed in I-2.However, minor changes in lymphocytes, including CD8+ T cells, were observed in II-3 and I-1.Interestingly, significant increases in IL-6 levels were observed in I-2 before the first recurrence, and IL-10 levels were high before the second, suggesting that high IL-6 or IL-10 levels may also predict the recurrence of SARS-Cov-2. Multiple scattered ground-glass shadows in the lung, a high viral load, and the decreased NLR provided clues that I-2 had developed severe pneumonia.A study has shown that monitoring the NLR is helpful in the early screening of critical illness and the diagnosis and treatment of COVID-19 (20).The condition of patients with CLL should be monitored according to the NLR rather than the absolute number of lymphocytes.Liu et al. (20) reported that the degree of lymphopenia and the severity of proinflammatory cytokine storms are increased in severe COVID-19 patients compared with those in patients with mild cases.Severe patients showed significant and sustained decreases in lymphocyte and T-cell counts and increases in IL-6, IL-10, IL-2, and IFN-γ levels, while mild cases exhibited increased neutrophil counts (12,21).The clinical characteristics in our case were consistent with the features of severe cases. Owing to humoral immune dysfunction, complete antibodies are improperly formed.Based on the inhibition of innate immune surveillance and the dysfunction of acquired immunity, clearance of the virus showed a delay in I-2 in comparison with I-1 and II-3.Complete antibodies were not formed, and weakened cell-killing effects prevented the virus from being completely cleared and led to repeated recurrence.Recurrent positivity for SARS-Cov-2 will likely occur in I-2 for a longer period of time.High-dose IVIG combined with antiviral therapy seems to be an effective treatment to alter the inflammatory response and prevent the development of severe pneumonia. ",
"section_name": "DISCUSSION",
"section_num": null
},
{
"section_content": "In CLL patients, a neoplastic antigen-specific B-cell clone proliferates and outgrows other B cells, leading to immune deficiency.Considering the low humoral immune response and ineffective clearance of SARS-Cov-2 in CLL patients, the follow-up and home quarantine period should be extended for such patients.The treatment of CLL may lead to further weakening of humoral immunity.Therefore, we need further studies to clarify whether CLL therapy should be suspended or continued during COVID infection.For those patients who are prone to progression into a severe stage, administering humoral immunity therapies can help to prevent disease progression and quickly meet the cure criteria. ",
"section_name": "CONCLUSION",
"section_num": null
}
] |
[
{
"section_content": "The authors gratefully acknowledge the patients and thousands of unsung heroes in fighting against the epidemic of COVID-19. ",
"section_name": "ACKNOWLEDGMENTS",
"section_num": null
},
{
"section_content": "FUNDING The research was supported by the academician workstation of the Fourth Affiliated Hospital of the Zhejiang University School of Medicine (2019-2024) and by funding from the Public Technology Research Projects of Yiwu, China (2016S05), the Key Project of Jinhua Science and Technology Plan (2020XG-29), the Key Medical Discipline of Yiwu, China (Hematology, 2018-2020), the Key Medical Discipline of Jinhua, China (Hematology, 2019-2021), and the Project of Educational Commission of Zhejiang Province of China (Y201942124). ",
"section_name": "",
"section_num": ""
},
{
"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 studies involving human participants were reviewed and approved by the Ethics Committee of the Fourth Affiliated Hospital of Zhejiang University.The patients/participants provided their written informed consent to participate in this study.Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article, and for the publication of this case report. XY, XX, BL, and JH: performed the research and wrote the manuscript.WZ and YL: performed the experiments and analyzed the data.JW and XH: assisted with laboratory experiments and provided technical advice.JinJ and DC: contributed to patient samples and assisted with data collection.JieJ and JH: designed the study and assisted in manuscript preparation.All authors approved the manuscript for submission. 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": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "The studies involving human participants were reviewed and approved by the Ethics Committee of the Fourth Affiliated Hospital of Zhejiang University.The patients/participants provided their written informed consent to participate in this study.Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article, and for the publication of this case report. ",
"section_name": "ETHICS STATEMENT",
"section_num": null
},
{
"section_content": "XY, XX, BL, and JH: performed the research and wrote the manuscript.WZ and YL: performed the experiments and analyzed the data.JW and XH: assisted with laboratory experiments and provided technical advice.JinJ and DC: contributed to patient samples and assisted with data collection.JieJ and JH: designed the study and assisted in manuscript preparation.All authors approved the manuscript for submission. ",
"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
}
] |
10.1038/s41408-022-00721-6
|
Clinical outcomes in patients with chronic lymphocytic leukemia with disease progression on ibrutinib
|
<jats:title>Abstract</jats:title><jats:p>Patients with chronic lymphocytic leukemia (CLL) with disease progression on ibrutinib have worse outcomes compared to patients stopping ibrutinib due to toxicity. A better understanding of expected outcomes in these patients is necessary to establish a benchmark for evaluating novel agents currently available and in development. We evaluated outcomes of 144 patients with CLL treated at Mayo Clinic with 2018 iwCLL disease progression on ibrutinib. The median overall survival (OS) for the entire cohort was 25.5 months; it was 29.8 months and 8.3 months among patients with CLL progression (<jats:italic>n</jats:italic> = 104) and Richter transformation (<jats:italic>n</jats:italic> = 38), respectively. Longer OS was observed among patients with CLL progression who had received ibrutinib in the frontline compared to relapsed/refractory setting (not reached versus 28.5 months; <jats:italic>p</jats:italic> = 0.04), but was similar amongst patients treated with 1, 2, or ≥3 prior lines (18.5, 30.9, and 26.0 months, respectively, <jats:italic>p</jats:italic> = 0.24). Among patients with CLL disease progression on ibrutinib, OS was significantly longer when next-line treatment was chimeric antigen receptor T-cell therapy (median not reached) or venetoclax-based treatment (median 29.8 months) compared to other approved treatments, such as chemoimmunotherapy, phosphoinositide 3’-kinase inhibitors, and anti-CD20 monoclonal antibodies (9.1 months; <jats:italic>p</jats:italic> = 0.03). These findings suggest an unmet need for this growing patient population.</jats:p>
|
[
{
"section_content": "Ibrutinib has demonstrated long-term efficacy in relapsed/ refractory (median progression-free survival [PFS] 44.1 months) [1] and frontline (5-year PFS estimate 70%) [2] patient populations with chronic lymphocytic leukemia (CLL), leading a therapeutic renaissance of targeted therapies capable of more frequent durable responses among even high-risk patient populations (4year PFS among patients with TP53 alterations 79%) [3].Despite these excellent outcomes, the majority of patients eventually discontinue ibrutinib treatment; disease progression and toxicity being the most common reasons.Patients who stop ibrutinib for disease progression have worse PFS and overall survival (OS) compared to patients who stop ibrutinib because of toxicity [4][5][6].Alternative classes of targeted agents (e.g., BCL2 antagonists, phosphoinositide 3-kinase inhibitors [PI3Ki], and next-generation anti-CD20 monoclonal antibodies) are now readily available in the clinic and have shown promise in the management of patients with relapsed CLL [7][8][9].In addition, auspicious new drugs, including non-covalent Bruton tyrosine kinase inhibitors (BTKi) such as nemtabrutinib and pirtobrutinib, are in development with preliminary studies showing impressive efficacy in relapsed CLL after ibrutinib failure [10,11].Finally, cellular therapies (including CAR-T and allogeneic stem cell transplant) also represent important treatment options that need to be considered for this group of high-risk patients.A better understanding of expected clinical outcomes in patients with disease progression on ibrutinib is necessary to establish a benchmark for evaluating future studies related to the actual event and options for therapy.Here, we focus on outcomes after progression on ibrutinib in a large cohort of patients with CLL, reporting survival estimates with varied treatments, line-of-therapy settings, and patterns of progression. ",
"section_name": "INTRODUCTION",
"section_num": null
},
{
"section_content": "After IRB approval, we reviewed the medical records of patients with CLL who received ibrutinib therapy for CLL at a Mayo Clinic Cancer Center sites (Arizona, Florida, or Minnesota) between 4/2012-6/2021.Baseline relevant clinical characteristics, prior therapies, duration of ibrutinib treatment, and post-ibrutinib therapy were abstracted for all patients.Date of progression on ibrutinib therapy was ascertained by retrospective chart review and was documented according to the 2018 iwCLL guidelines [12].Resistance mutation sequencing was conducted at NeoGenomics reference laboratory; methods are included in the Supplemental Materials. Treatment-free survival (TFS) was analyzed as the duration from the start of treatment immediately after ibrutinib failure to the start of the subsequent line of therapy or death, whichever occurred earlier.Overall survival (OS) was analyzed as the time from date of progression while on ibrutinib and from subsequent therapy start date until date of death or last known to be alive.OS was analyzed using the Kaplan-Meier method (with comparisons of OS by characteristics analyzed by the log rank test) and Cox proportional hazards model.The association with OS and the event of venetoclax treatment at any time post-progression was analyzed as a timedependent covariate in Cox proportional hazards models.Statistical analyses were conducted using SAS 9.4. ",
"section_name": "METHODS",
"section_num": null
},
{
"section_content": "",
"section_name": "RESULTS",
"section_num": null
},
{
"section_content": "A total of 144 patients were identified who had progression of disease on ibrutinib therapy; 106 patients had progression of CLL, whereas 38 patients developed biopsy-proven Richter transformation (35 with diffuse large B-cell lymphoma [DLBCL], 3 with classical Hodgkin lymphoma).The characteristics of these 144 patients at the time of ibrutinib start as well as at the time of progression are shown in Table 1.The median age at the time of progression on ibrutinib was 68 years (range, 43-92).Ibrutinib was used as first-line therapy in 16% of patients.A total of 37/54 (69%) assessed patients had BTK/PLCG2 mutations identified (19 BTK mutation only; 9 PLCG2 mutation only; 9 both BTK and PLCG2 mutations); 34/45 (76%) in patients with CLL progression and 3/9 (33%) in patients who experienced Richter transformation. ",
"section_name": "Patients and disease characteristics",
"section_num": null
},
{
"section_content": "The median OS of the entire cohort after progression on ibrutinib was 25.5 months (95% CI 17.7-31.0).Not unexpectedly, the OS was significantly different between those who experienced CLL disease progression versus Richter transformation (median 29.8 versus 8.3 months, respectively; p = 0.002, Fig. 1).The median OS of patients who experienced CLL disease progression when ibrutinib was used in the first-line setting was longer compared to those treated in the relapsed/refractory setting (not reached versus 28.5 months; p = 0.04; Fig. 2A).The median follow-up of patients from time of progression was 16.6 months overall; 23.5 months among patients treated in the first-line setting and 15.9 months in the relapsed/refractory setting.Among patients treated in the relapsed/refractory setting, the median OS when ibrutinib was used after one prior line (n = 20), 2 prior lines monoclonal antibody) and 18 patients received venetoclax plus continued ibrutinib (±anti-CD20 monoclonal antibody).Patients who continued ibrutinib with venetoclax-based treatment had similar TFS to those who did not continue ibrutinib (median 23.7 versus 16.7 months; p = 0.26; Fig. 6).TP53 disruption was not associated with TFS (HR 1.2, 95% CI 0.7-2.1;p = 0.51). ",
"section_name": "Survival outcomes after progression on ibrutinib among the overall cohort",
"section_num": null
},
{
"section_content": "In univariate analyses, IGHV mutation status, TP53 disruption, and the presence of a BTK or PLCG2 mutation status were not predictors of shorter OS from time of CLL disease progression on ibrutinib.Time from iwCLL progression to start of subsequent therapy ≥1.5 months versus <1.5 months was associated with longer OS from subsequent therapy (47.1 versus 25.6 months; p = 0.03). Treatment outcomes in patients with Richter transformation Among the 35 patients who had transformation to DLBCL, the most common first salvage therapy consisted of chemoimmunotherapy in 15 patients, immune checkpoint inhibitor therapy in nine patients, venetoclax-based therapy in three patients, and PI3Ki-based treatment, anti-CD20 monoclonal antibody treatment, and antibody drug conjugate treatment in two patients each.patients died before any salvage therapy could be administered because of progressive disease.The OS and TFS of these patients according to the types of treatments administered is shown in Supplemental Figs.2A and2B, respectively.Type of treatment did not have a significant impact on OS nor TFS. Among the three patients who had transformation to Hodgkin lymphoma, two remained alive at last follow-up >4 years after receipt of ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) with no relapse of Hodgkin lymphoma.One patient died amidst neutropenic infection while in a partial remission after treatment with BCVPP (carmustine, vinblastine, cyclophosphamide, procarbazine, prednisone). ",
"section_name": "Clinical presentation and outcomes in patients with CLL progression",
"section_num": null
},
{
"section_content": "Here, we report on a meaningfully large cohort of nearly 150 patients showing a median overall survival of 25.5 months from the time of iwCLL progression on ibrutinib.This is strikingly similar to that reported in a recent abstract from The Ohio State University group on a similar cohort (median 24.4 months) [13].Outcomes beyond CLL progression on ibrutinib were similar among those treated in the relapsed/refractory setting irrespective of number of prior lines, but differed by immediate subsequent therapy, favoring venetoclax-based and CAR T treatments.clinical observations at the time of progression, including pattern of progression and time from progression to next therapy start, also showed prognostic OS relevance in our study.These data improve our current understanding of outcomes in this growing patient population and provide a critical benchmark when considering trials of promising novel agents in development.Treatment patterns and outcomes following disease progression on a BTKi among CLL patients are less well represented in the randomized, prospective clinical trials of approved treatments in the relapsed/refractory space [7,8].Extrapolating predictions for patients with ibrutinib-refractory disease from data in ibrutinibexposed patients is problematic and therapeutic decisions are guided by single-arm prospective studies, subgroup analyses, and limited retrospective cohorts. Previous studies have shown a limited benefit to be expected with PI3Ki or chemoimmunotherapy (median PFS 9 months and 5.1 months, respectively) in patients who previously received ibrutinib and stopped for any reason [14].Our study demonstrates patients with CLL disease progression on ibrutinib have better survival outcomes with venetoclax-based regimens compared to other approved options (median OS 29.8 versus 9.1 months; median TFS 20.1 versus 4.4 months).In the prospective study of venetoclax monotherapy post-BTKi treatment, the median PFS was 24.7 months and 12-month OS rate 91%, without separating that cohort by exposed and refractory subgroups.Overall response was higher among patients non-refractory to prior BTKi treatment (63% versus 54%) [15].A pooled analysis of these patients and others treated with venetoclax on a clinical trial post-BTKi reported an ORR of ~65% without differentiating BTKiexposed versus -refractory [16].In our cohort, venetoclax at any time post-ibrutinib progression did not impart the same OS benefit observed when analyzing next-line venetoclax compared to alternative options.While difficult to reconcile these findings entirely, comparing outcomes pre-and post-venetoclax approval date also hints at an importance in sequence of therapies.Altogether, our new results and these previously reported data support the most common current practice of proceeding to venetoclax-based treatment as next-line treatment following progression on ibrutinib in venetoclax-naïve patients.CAR T also has therapeutic efficacy in highly refractory patients but lacks current approval [17].Patients treated with CAR T in our cohort had outstanding outcomes (illustrated by a median OS not reached with no events among the six patients), but conclusions are precluded by the limited number of patients and the optimal timing to pursue cellular therapy in the novel agent era remains unknown. Considering the risk for disease flare with ibrutinib interruption, particularly amidst progressive disease, the synergistic combination of ibrutinib and venetoclax has appeal in the post-ibrutinib progression setting as well [18].However, when focusing on the venetoclax-based treatment subgroups in this study, no significant difference in TFS was observed with continued ibrutinib.Results have not yet been reported for a prospective trial (NCT03422393) evaluating dose-escalated ibrutinib and the addition of venetoclax for next-line treatment at ibrutinib progression. We demonstrated the pattern of progression, specifically progressive lymphadenopathy, was associated with postprogression OS.Median survival estimates among the pattern of progression subgroups (17.7 months and 46.7 months for patients with lymphadenopathy without lymphocytosis and lymphocytosis without lymphadenopathy, respectively) closely resembled those presented by the OSU group (15.2 months and 49.9 months for the same groups) [13].Better understanding this difference is an area of active research.A possible contribution could be differing mechanisms of resistance.Resistance mutations in BTK and PLGC2 were less frequently detected among patients relapsing with lymphadenopathy (40%) compared to those with lymphocytosis (81%) in a prior study [19].Another potential reflection of varied CLL biology at relapse on ibrutinib is our finding that patients who received subsequent treatment ≥1.5 months beyond relapse had OS that was approximately twice as long as patients receiving next-line therapy sooner.One possible reason for this apparent paradox is that the patients progressing in a more gradual, less dramatic fashion and thus in less need of urgent change in therapy are biased to more favorable outcomes, similar to the diagnosis-to-treatment interval shown in patients with newly diagnosed large cell lymphoma [20]. Survival following Richter transformation to DLBCL on ibrutinib was similarly dismal whether occurring in the frontline or relapsed/refractory setting, emphasizing the continuing urgent need for better Richter transformation treatments.Outcomes differed between frontline and relapsed settings in those with CLL progression with longer survival observed following progression on frontline ibrutinib.Unexpectedly, among patients who received ibrutinib in the relapsed setting, post-progression survival was similar across patients with one to three or more prior lines of therapy.This finding seems to place emphasis on treatment after progression on ibrutinib but requires validation in independent cohorts. The majority of ibrutinib treatment in this study occurred in the relapsed/refractory setting, which may be less reflective of contemporary practice and is a limitation of the study.Our experience here is that of a tertiary referral center, which likely explains the higher-than-expected number of Richter transformation cases observed and should not be interpreted as a true incidence rate.Exploring the impact of resistance mutations in outcomes and patterns of progression is limited due to lack of consistent sequencing in this cohort.Similarly, the limitation of non-uniform follow-up inherent to a retrospective study precludes reporting a reliable response assessment.However, strengths include (1) our focus on meaningful TFS and OS outcomes; (2) description of clinical features of the disease progression while on ibrutinib made possible by a well-annotated, prospectively maintained CLL Database; (3) relatively long follow-up compared to earlier studies evaluating this patient population.These aspects of the study facilitate providing greater insight overall into outcomes of CLL patients in the post-ibrutinib progression period. Results from this study demonstrate that progression of disease on ibrutinib represents an ongoing unmet need in patients with CLL.Although non-covalent BTKi, such as nemtabrutinib and pirtobrutinib, show promising efficacy in this setting, they currently lack label approval and are only available through a clinical trial [10,11].Participation in well-designed clinical trials is remains key for this growing patient population.Venetoclax-based treatments (if not given before) are the current standard in the clinic for this group and indeed offered amongst the best TFS and OS outcomes in this study.We speculate that the role for continued ibrutinib beyond progression in combination with venetoclax in certain patients and the impact and optimal timing of cellular therapy remain important questions. ",
"section_name": "DISCUSSION",
"section_num": null
}
] |
[
{
"section_content": "The conduct of this research was supported in part by the Henry J. Predolin Foundation. ",
"section_name": "ACKNOWLEDGEMENTS",
"section_num": null
},
{
"section_content": "Data not available without request and IRB review due to patient confidentiality. ",
"section_name": "DATA AVAILABILITY",
"section_num": null
},
{
"section_content": "PJH, KGR, and SAP designed the research, collected, analyzed, and interpreted data, and wrote the manuscript; TGC, WD, JFL, AAC-K, SSK, EM, YW, SA, ABK, RP, TS, CAH, MS, DLVD, EB, and NEK cared for the patients, interpreted the results, and critically reviewed/ revised the manuscript; SMS acquired data and critically reviewed/resvied the manuscript; SLS and KGR analyzed data, conducted statistical analyses, and critically reviewed/revised the manuscript; all authors approved the manuscript in its final format. The authors declare no competing financial interests in direct relation to the work reported.The following authors also declare no competing financial interests otherwise: PJH, KGR, TGC, JFL, RP, SMS, TS, CAH, MS, DLVD, EB, SLS,.In the interest of transparency, additional potential conflicts of interest for the remaining authors are included subsequently.WD: Research funding has been provided to the institution from Merck The online version contains supplementary material available at https://doi.org/10.1038/s41408-022-00721-6. Correspondence and requests for materials should be addressed to Sameer A. Parikh. Reprints and permission information is available at http://www.nature.com/reprintsPublisher'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": "PJH, KGR, and SAP designed the research, collected, analyzed, and interpreted data, and wrote the manuscript; TGC, WD, JFL, AAC-K, SSK, EM, YW, SA, ABK, RP, TS, CAH, MS, DLVD, EB, and NEK cared for the patients, interpreted the results, and critically reviewed/ revised the manuscript; SMS acquired data and critically reviewed/resvied the manuscript; SLS and KGR analyzed data, conducted statistical analyses, and critically reviewed/revised the manuscript; all authors approved the manuscript in its final format. ",
"section_name": "AUTHOR CONTRIBUTIONS",
"section_num": null
},
{
"section_content": "The authors declare no competing financial interests in direct relation to the work reported.The following authors also declare no competing financial interests otherwise: PJH, KGR, TGC, JFL, RP, SMS, TS, CAH, MS, DLVD, EB, SLS,.In the interest of transparency, additional potential conflicts of interest for the remaining authors are included subsequently.WD: Research funding has been provided to the institution from Merck ",
"section_name": "COMPETING INTERESTS",
"section_num": null
},
{
"section_content": "The online version contains supplementary material available at https://doi.org/10.1038/s41408-022-00721-6. Correspondence and requests for materials should be addressed to Sameer A. Parikh. Reprints and permission information is available at http://www.nature.com/reprintsPublisher'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
}
] |
10.3390/ijerph18158193
|
Changes of Dentition State in Leukemic Patients during Chemotherapy
|
<jats:p>A number of systemic diseases including hematological disorders have manifestations in the oral cavity region. These manifestations may often represent early signs of the underlying hematopoietic disease and occur frequently in leukemia. Despite the fact that leukemia has long been known to be associated with oral health deterioration, the available literature on this topic consists mostly of case reports, without data to conclude these. The aim of the study was to assess dentition state in leukemic patients during one cycle of chemotherapy and its correlation with blood parameters. The study included 102 adults treated because of leukemia at the Clinic of Haemato-Oncology and Bone Marrow Transplantation at the university hospital in Lublin, Poland. The sample group consisted of 51 women and 51 men aged 22 to 72 (54.07 ± 10.33) with following diagnoses: Acute myelogenous leukemia (AML)—55 patients (53.92%), Chronic lymphocytic leukemia (CLL)—17 patients (16.67%), Acute lymphoblastic leukemia (ALL)—16 patients (15.69%), Chronic myelogenous leukemia (CML)—10 patients (9.80%), Acute promyelocytic leukemia (APL) —3 patients (2.94%), Chronic hairy cell leukemia (HCL)—1 patient (0.98%). DMFT index was used to assess dentition state. After the cycle of chemotherapy, their dentition state changed in terms of decayed, missing and filled teeth and correlated with hematological parameters. Adult patients with leukemia have high dental treatment needs, and high number of missing teeth; thus, a comprehensive and fast dental treatment is necessary to avoid systemic complications and ensure better quality of life.</jats:p>
|
[
{
"section_content": "A number of systemic diseases including hematological disorders have manifestations in the oral cavity region.These manifestations may often represent early signs of the underlying hematopoietic disease and occur frequently in leukemia.The symptoms may point out the initial evidence of the disease. Leukemia (Lat.leukemia) is a neoplastic disease originating in the hematopoietic system in which all morphotic elements of the blood are formed.It is characterized by an unlimited and irreversible proliferation of immature hematopoietic or lymphatic cells.The proliferation of these cells can occur both in sites typical for hematopoietic activity, as well as in other tissues and organs.These diseases manifest as quantitative and qualitative changes in leukocytes in the bone marrow, spleen, blood and lymph nodes.The changes occur as a result of neoplastic transformation of stem cells originating from the early stages of hematopoiesis.Clinically, as with other hematopoietic and lymphatic system diseases (bone marrow aplasia, lymphomas and others) hemorrhagic diathesis, granulocytopenia, anemia and impaired immunity may occur.Leukemia is more common in men than in women (3:2), and individual types show the maximum incidence at different ages.General division of leukemias may be presented as follows: acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML) and chronic lymphocytic leukemia (CLL).The classification was modified in the course of the development of knowledge about morphology and features of cancer cells.Nowadays, the classification of hematopoietic neoplasms is based on the 2016 WHO classification [1][2][3][4]. Acute leukemia has two incidence peaks: in adolescence and older age, for chronic leukemia, the median age is about 50 years, while in children it is rare.The standardized death rate due to different types of leukemia is about 7 in 100,000 inhabitants; there are race differences and the level of harmful factors, including environmental factors.The cause of leukemia is usually unknown.The exceptions are acute hyperplasia in people exposed to ionizing radiation and some cytostatics-these leukemias are called therapy related AML (t/AML).Cytostatics that contribute to t/AML are alkylating drugs and topoisomerase II inhibitors.In about 20% of leukemia cases, the disease is detected by chance (peripheral blood counts taken for various reasons).Some patients develop general symptoms.Other symptoms are related to anemia or thrombocytopenia.Hyperviscosity syndrome caused by high leukocytosis may develop.The symptoms of hyperviscosity syndrome are the onset or worsening of heart failure, hypertension, disturbances of consciousness, including coma.These symptoms result from impaired oxygenation of vital organs.There is also a vascular diathesis.Abdominal pain due to enlargement of the liver and/or spleen, bone pain may occur.The most common clinical signs of leukemia are weight loss, fever, anemia, hemorrhage, hepatosplenomegaly and lymphadenopathy.In addition to the lymphatic system, neoplastic cells are located in the bones, bone marrow and central nervous system, giving clinical signs from these tissues and organs [5][6][7][8]. Clinically, leukemia is manifested by oral symptoms, and often the first symptom of the disease is bleeding gums and petechiae within the oral mucosa.Other symptoms may include inflammatory and specific infiltrates, erosions of the oral mucosa, tongue pain and burning, ulcers and necrotic lesions, swelling and gingival hyperplasia.Patients have impaired healing due to reduced anti-inflammatory defense, and chemotherapy can cause clinical changes in the periodontium and mucosa, often difficult to distinguish from the underlying disease.Chemotherapy also contributes to secondary bacterial, viral and fungal infections.After chemotherapy, general symptoms such as permanent cervical lymphadenopathy, malaise, pallor because of anemia or ulcerations related to the immune deficiency may occur.The listed symptoms may be accompanied by gingival bleedings, bleedings from the nose, petechiae, general weakness, malaise and mild fever.Therefore dentists/ oral medicine specialists play an important role in therapy of patients presenting these common symptoms both of the disease itself and its treatment [9][10][11][12][13]. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "Presence of pathological changes in soft tissues of the oral cavity among patients with leukemia is well-documented.The data on the state of these patients' dentition, which is also important in oral health, is still limited.The present study aimed to assess the dentition state in leukemic patients during one cycle of chemotherapy. ",
"section_name": "AIM",
"section_num": null
},
{
"section_content": "",
"section_name": "Materials and Methods",
"section_num": "2."
},
{
"section_content": "The study included an accessible sample of leukemic patients being treated with chemotherapy for different types of leukemia in the Clinic of Haemato-Oncology and Bone Marrow Transplantation of the Independent Public Clinical Hospital No. 1 in Lublin, Poland.This hospital is a reference unit for leukemia treatment of adults in the lubelskie voivodeship (region). Exclusion criteria were as follows: patients under the age of 18 years old, patients receiving stem cell transplantation or radiotherapy or suffering from hematological diseases other than leukemia.Patients who started palliative health care or whom general state made dental examination impossible were not eligible for the study. Initial and following dental examinations were performed by the same examiner-a dental practitioner, with 5 years of clinical experience.The examiner received appropriate training and was calibrated by an experienced clinician, a dentist too.Intraexaminer reproducibility was measured for this examiner and proved to be sufficient to perform the study. The study included 51 women and 51 men aged 22 to 72 (54.07 ± 10.33).The time that has elapsed since the patients was diagnosed was between 1 and 10 years with following diagnoses: Acute myelogenous leukemia (AML)-55 patients (53.92%),Chronic lymphocytic leukemia (CLL)-17 patients (16.67%),Acute lymphoblastic leukemia (ALL)-16 patients (15.69%),Chronic myelogenous leukemia (CML)-10 patients (9.80%), Acute promyelocytic leukemia (APL)-3 patients (2.94%), Chronic hairy cell leukemia (HCL)-1 patient (0.98%).The simplified division of leukemia into its chronic and the acute forms was used for statistical analysis.In 28 patients (27, 45%), chronic leukemia was stated; acute leukemia was diagnosed in 74 patients (72, 55%).All clinical data of patients, including laboratory test results, information about the applied treatment and its course, were obtained from hospital records.All participants provided written informed consent prior to enrollment in the study. ",
"section_name": "Participant Selection",
"section_num": "2.1."
},
{
"section_content": "The initial examination, in which dentition state was assessed, was carried out before the next phase of leukemia treatment, e.g., before the next cycle of chemotherapy, right after medical qualification for the chemotherapy cycle.The following study was conducted after the chemotherapy cycle had been completed (after 7-14 days, depending on type of leukemia and the treatment protocol). Examination took place at the hospital conditions, utilizing head torch and compressed air can.A basic dental diagnostic kit and a WHO 621 probe was used.Dental index (DMFT), commonly used in the clinical diagnostics, was calculated and used in accordance with WHO criteria [14] to evaluate the state of dentition.The DMFT index comprised decayed (D), missing (M), and filled or crowned (F) teeth (T) and added them to a final sum (possible range 0-32).A decayed tooth (D) and cavitated caries lesions were recorded when the tooth surface had an unmistakable cavity, detectably softened wall or undermined enamel (including secondary caries and temporary fillings).Initial caries (white spot lesions) were not considered in the present study.Restorations were recorded as \"F\" and missing teeth as \"M\" only when applied to caries-related reasons.Other enamel defects like fluorosis, amylogenesis imperfecta were not registered.All procedures performed in the study were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. ",
"section_name": "Dentition State Assessment",
"section_num": "2.2."
},
{
"section_content": "The statistical analysis was carried out with the Statistica 12 software package (StatSoft, Tulsa, OK, USA).The calculation for quantitative traits included range of values (min., max), arithmetic mean (M), and standard deviation (SD).Differences between the compared groups for quantitative traits were verified with tests (depending on the stated distribution): for dependent variables-Students'-t, Wilcoxon, for independent variables-Student's-t, Mann-Whitney U test.The differences in the prevalence of the analyzed traits between particular groups were tested with χ 2 test (Fisher's χ 2 ) test).The study adopted a 5% risk of error; therefore, statistically significant differences were those of p < 0.05. ",
"section_name": "Statistical Analysis",
"section_num": "2.3."
},
{
"section_content": "",
"section_name": "Results",
"section_num": "3."
},
{
"section_content": "The mean DMFT value in leukemic patients before and after hematological treatment was 24.00 ± 6.17.There was no statistically significant change in the DMFT index after chemotherapy cycle.Dental caries incidence expressed as D number equaled 0 to 17 both at the first and the second appointment.The mean D index in the first examination was 4.48 ± 4.65, whereas after the treatment it was 4.06 ± 4.49.The reduction in D value after hematological treatment was statistically significant.The index of missing teeth (M) was 16.01 ± 9.81 in the first examination and 16.26 ± 9.78 in the follow-up examination and the change in its value was statistically significant.The mean number of teeth with fillings (F) was 3.51 ± 4.55 and 3.68 ± 4.68 in the first and second examinations, respectively.The change in those numbers was statistically significant (Table 1).Based on the difference between the values in hematological parameters after the chemotherapy cycle and the values before the treatment-patients were divided into two groups: 1. Patients with lower or unchanged hematological parameters (↓) after treatment; 2. Patients with higher hematological parameters (↑) after treatment. DMFT index and its components (D, M, F) were analyzed depending on the direction of changes in hematological parameters (↓ vs. ↑). ",
"section_name": "Chemotherapy Cycle Versus DMFT Index",
"section_num": "3.1."
},
{
"section_content": "No statistically significant difference was found for DMFT index depending on the direction of change in the platelet count in patients after hematological treatment.Significantly higher values of the caries index (D) were found both in the first and in the second study in patients whose platelet count increased after chemotherapy (Table 2).No statistically significant difference was found for DMFT index depending on the direction of change in the white blood cells count in patients both before and after chemotherapy cycle.Statistically significantly higher values of the caries index D (decayed) were observed in patients with white blood cells count increase both before and after chemotherapy (Table 3).The difference in DMFT value was statistically significant depending on the direction of change of neutrophils in the studied patients, both before and after the chemotherapy.The change in the values of D (decayed) and M (missing) indices was also statistically significant (Table 4).Both before and after chemotherapy, the change in the number of lymphocytes did not correlate with the significant change of DMFT index.On the other hand, a significantly higher D index was found in patients whose lymphocyte count increased after the treatment (Table 5).The change in the number of monocytes correlated with a significant change of the values of indicator-both before and after treatment-DMFT, D, M (Table 6).When assessing the change in the number of eosinophils, a significant difference was found in the DMFT value in patients after the applied hematological treatment.The mean DMFT value in people with decreased eosinophil counts was 24.60 ± 6.23, while in patients with an increase in eosinophils, the mean DMFT was 22.04 ± 5.66 (Table 7).In patients with an increased number of basophils, a significantly higher value of F (filled) was found before and after chemotherapy (Table 8). ",
"section_name": "Platelets (PLT)",
"section_num": "3.2.1."
},
{
"section_content": "There was no significant difference in DMFT index between patient groups both before and after chemotherapy regardless of the direction of change in red blood cells (Table 9). ",
"section_name": "Red Blood Cells (RBC)",
"section_num": "3.2.8."
},
{
"section_content": "DMFT index was also assessed depending on the type of leukemia.In patients with chronic leukemia, a significantly higher value of F index (filled teeth) was found compared to patients with acute leukemia, both before and after chemotherapy (Table 10).Depending on the duration of the disease, the values of DMFT index was assessed.A significant correlation between the disease duration was found on the DMFT index values before and after hematological treatment.The mean DMFT value was higher in people with longer duration of the disease and was 26.00 ± 5.78 (Table 11).The correlation between patients' age on DMFT values was assessed.A statistically significantly higher M number value was found before and after hematological treatment in older patients.However, a statistically significantly higher value of F number before and after chemotherapy was found in younger patients (Table 12). ",
"section_name": "Type of Leukemia in Relation to Changes in Dentition",
"section_num": "3.3."
},
{
"section_content": "There was no statistically significant difference in the values of DMFT index depending on the gender of patients, neither before nor after hematological treatment (Table 13). ",
"section_name": "Gender and the Condition of the Oral Cavity",
"section_num": "3.6."
},
{
"section_content": "The studies examining the dentition in adult patients with leukemia are rare, and up until now, the data of the oral health situation has been insufficient.Oral manifestations and initial symptoms are often important in the early detection of leukemia; in approximately 25% of the patients with AML, dentists are involved in the diagnosis.Apart from this, poor oral health is associated with a higher incidence of systemic infections and complications in patients suffering from leukemia.Accordingly, appropriate dental therapy before chemotherapy or stem cell transplantation can decrease the rate of septicemia cases and its fatal consequences [7,[15][16][17][18][19]. In interpreting results of the present study, the low number of available data has to be considered.While there are several publications on the oral health status of children suffering from leukemia [14,17,[20][21][22], not many investigations are focused on adult patients.Recent papers primarily describe case studies or retrospective studies with focus on AML and ALL and are not able to illustrate the oral health situation of patients before and after chemotherapy in different types of leukemia [23][24][25][26]. Busjan et al. conducted their research on 39 patients newly diagnosed with acute leukemia (ALL and AML) and 38 control patients that were matched to the leukemic patients by age, gender and smoking habits.The dental examination of the study group took place a few days after they were diagnosed-before beginning any treatment of the disease.The researchers claimed that there was no significant difference in DMFT values between the leukemic (combined ALL and AML) patients and control group.(18.69 ± 6.38 vs. 16.62 ± 7.4).Moreover, no significant difference in DMFT scores was found between patients suffering from AML and ALL (19.54 ± 5.06 vs. 17.00 ± 8.43).In our study, patients with leukemia (all types) both before and after the chemotherapy cycle presented mean DMFT index value of 24.00 ± 6.17.Moreover, we did not find any significant difference between DMFT values of 74 patients with acute and 28 patients with chronic leukemia (24.03 ± 5.85 vs. 23.93 ± 7.07) neither before nor after the treatment.By analyzing individual components of the index, Busjan et al. found significantly higher D and M values in patients with leukemia than in control groups.The mean age of patients in both studies was similar (55.61 ± 17.01 vs. 54.07 ± 10.33 in our study) [25]. According to Meyer et al., who conducted a study on immunocompromised patients, no statistically significant differences were found in DMFT index scores between groups of patients with leukemia-not in AML or ALL (53 patients, with either AML or ALL, mean DMFT 18.8 ± 9.0), systemic lupus erythematosus, heart transplant recipients or the control group.Presented value of mean DMFT index was similar to the research conducted by Busjan et al. [26]. In our study, the mean M (missing teeth) value of leukemic patients was 16.01 ± 9.81 before and 16.26 ± 9.78 after chemotherapy cycle.Comparing this result with the general population of different countries in this age group (45-64 years), it can be seen that patients from the present study on average have more teeth missing than people from a similar age group without this diagnosis.Suffering from leukemia may be considered one of the reasons of this situation.For example, in 855 citizens of Georgia aged 45-64, mean M value was 6.53 ± 6.47 [27], and 1945 Hungarian citizens from the general population who participated in the study had M mean value of 9.07 ± 7.24 [28]. According to the latest data of the Polish Ministry of Health, mean M index value in age group 35-44 living in a city was 3.1 ± 4.0 in 2010 and 1.2 ± 2.0 in 2017.Mean M value for Lublin voivodeship in this age group was 10.4 ± 4.4 in 2010 and 3.7 ± 1.8 in 2017.For age group 65-74 living in a city, the value of M index was 21.4 ± 7.9 in 2009 and 13.9 ± 8.8 in 2019.Mean M value for Lublin voivodeship for 2009 is missing, while in 2019 it was 12.5 ± 7.2.Data for population aged 45-64 is lacking [29]. Consequently, knowledge of the oral conditions of patients is insufficient, and the need of treatment for diagnosed patients appears unclear.Data from the US National Cancer Institute claims that some cancer centers encourage tooth brushing and flossing, while others indicate the interruption of brushing and flossing when blood components have a drop below specified limits (e.g., platelets < 30,000 cells/mm 3 ).However, according to the institute itself, there is no evidence in the literature regarding the best approach.The centers providing strategy argue that the benefits of proper brushing and proper flossing outweigh the risks because the interruption of routine oral hygiene increases the risk of infection, and this could promote bleeding as well as increase the risk of local and systemic infection [7,30].There is an increased risk of severe infections due to immunosuppression accompanying underlying disease and its treatment in patients with leukemia.In this respect, bacteremia from various sources is an important problem.Diseases of the oral cavity, especially decayed teeth and periodontitis, are associated with the development of bacteria with high pathological potential.That factor allows the entry of a large number of bacteria into the blood circulation, even during daily routine procedures such as oral hygiene or chewing.It has been found that the high incidence of gingivitis or periodontitis is an independent risk factor for infectious complications during chemotherapy [31][32][33].In addition, dental interventions, especially oral surgery during chemotherapy, can have serious consequences such as bleeding complications.Therefore, it is recommended to perform dental clearance before induction chemotherapy to avoid both infectious complications and the need for dental intervention during therapy [34].This is also confirmed by the study of dental clinicians, in which dental rehabilitation has priority over induction therapy [35].According to these recommendations, the oral mucosa, dentition, periodontium should be carefully examined before chemotherapy. Diagnostic and therapeutic difficulties in the oral cavity are additionally caused by exacerbations and remissions of the general disease.Maintaining proper oral hygiene by the patient may be difficult due to pain, intense bleeding and ulcerations often occurring after chemotherapy.It is necessary to thoroughly cleanse the mouth and use rinses to inhibit the growth of micro-organisms to reduce the severity of the disease symptoms and side effects of therapy.Treatment should lead to a reduction in pain, dry mouth and promote healing of necrotic changes.So far, no universal way to prevent or treat inflammation of the periodontium has been found in patients undergoing chemotherapy.The use of rinses containing chlorhexidine in this group of patients did not bring the expected results.Clinical observations show that salivary substitutes, rinsing the mouth with sterile water, irrigations with sodium bicarbonate and the use of local anesthetics are effective [35][36][37][38]. In this study, statistically significant changes in D, M, F values resulted from restorative procedures (treatment of carious cavities) or tooth extractions.Therefore, awareness of the importance of dental treatment along with the medical management of these patients should be spread.A dental surgeon may be the first doctor to see a patient with hematopoietic and lymphatic disorders.It is very important to assess the changes in the mouth of a patient who is already undergoing treatment for a hematological disease [25,[39][40][41]. The study is limited by the fact that dental examination was not performed at the dental office condition.Moreover, examined patients are people with poor general health and compromised dental hygiene.Population of patients being treated in this hospital consist generally of people from the voivodeship and cannot be extrapolated to the whole country, and only patients over 22 years of age have been considered. However, the strength of present study is the sample size, which is relatively high for the studies focused on similar subject. Further studies are necessary to fully understand the causes of tooth loss in leukemic patients and provide solutions to prevent it. ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "1. On the basis of the obtained values of dental indices (D, M, F, DMFT), it was observed that patients' dentition state changed after the cycle of chemotherapy that was used. 2. The change in hematological parameters (apart from RBC) after chemotherapy correlated with change in DMFT index and/or its components. 3. The high prevalence of dental caries confirms the need for early and consistent dental treatment of patients with leukemia, especially considering hematological therapy. 4. Patients suffering from leukemia require permanent dental control.It can mitigate pathological processes in the oral cavity related with the disease and its treatment.Such management will allow the prevention of local complications such as tooth loss and will also affect patients' general state. ",
"section_name": "Conclusions",
"section_num": "5."
}
] |
[
{
"section_content": "Data Availability Statement: Not applicable. The authors declare no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "Data Availability Statement: Not applicable. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors declare no conflict of interest. ",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. ",
"section_name": "",
"section_num": ""
}
] |
10.3390/cancers12123664
|
Insights on TAM Formation from a Boolean Model of Macrophage Polarization Based on In Vitro Studies
|
<jats:p>The tumour microenvironment is the surrounding of a tumour, including blood vessels, fibroblasts, signaling molecules, the extracellular matrix and immune cells, especially neutrophils and monocyte-derived macrophages. In a tumour setting, macrophages encompass a spectrum between a tumour-suppressive (M1) or tumour-promoting (M2) state. The biology of macrophages found in tumours (Tumour Associated Macrophages) remains unclear, but understanding their impact on tumour progression is highly important. In this paper, we perform a comprehensive analysis of a macrophage polarization network, following two lines of enquiry: (i) we reconstruct the macrophage polarization network based on literature, extending it to include important stimuli in a tumour setting, and (ii) we build a dynamical model able to reproduce macrophage polarization in the presence of different stimuli, including the contact with cancer cells. Our simulations recapitulate the documented macrophage phenotypes and their dependencies on specific receptors and transcription factors, while also unravelling the formation of a special type of tumour associated macrophages in an in vitro model of chronic lymphocytic leukaemia. This model constitutes the first step towards elucidating the cross-talk between immune and cancer cells inside tumours, with the ultimate goal of identifying new therapeutic targets that could control the formation of tumour associated macrophages in patients.</jats:p>
|
[
{
"section_content": "As all living cells, macrophages perceive and respond to intra-and extracellular signals in order to maintain their functions (endocytic, phagocytic and secretory, for example) by displaying a wide spectrum of specific phenotypes (polarizations) in different inducer environments.Based on their activity and the expression of specific proteins, markers and chemokines, two major subsets of macrophages have been identified, namely classically activated macrophages (M1) exhibiting a pro-inflammatory response, and alternatively activated macrophages (M2, themselves subdivided into 4 subclasses: M2a, M2b, M2c, M2d [1][2][3]) exhibiting an anti-inflammatory response.Additionally, multiple studies support the idea that M1 and M2 macrophages represent, in fact, the extremes of a continuous polarization spectrum of cells deriving from the differentiation of monocytes [4].Their plastic gene expression profile is determined by the type, concentration and duration of exposure to the polarization stimuli in an inflammatory environment [3,[5][6][7][8]. Macrophages are also found inside tumors, as part of the tumor micro-environment (TME), a complex collection of cells that are found surrounding cancer cells including also other immune cells, such as lymphocytes and neutrophils, as well as other normal cells.In many tumors, infiltrated macrophages display mostly an M2-like phenotype, which provides an immunosuppressive microenvironment.In cancer, these tumor associated macrophages (TAMs) secrete several cytokines, chemokines and proteins which promote tumor angiogenesis, growth and metastasis [9][10][11][12].Interestingly, it was observed that in established tumors, signals originating from cancer cells can cause phenotypic shifts in macrophages, leading to alternative functions that do not correspond to either M1 or M2 phenotypes [13].Several studies have demonstrated that TAMs directly suppress CD8 + T cell activation in vitro [14][15][16][17].Mechanisms that orchestrate this process, either directly or indirectly, remain unclear [18] and warrant further exploration due to macrophages' important impact on tumor progression. The TME can be defined as an ecological system given the presence of diverse types of cells that interact in specific ways with each other.To name a few, cytotoxic T lymphocytes can attack cancer cells and kill them, while macrophages can phagocyte apoptotic or dead cells.However, complex feedback relations exist between the signals produced by some cell types and the phenotypic transitions that are induced by these signals in other cells.For example, due to the high density of leukemic B cells, monocytes residing in lymph nodes from CLL patients tend to differentiate into a pro-tumoral state, which is able in turn to promote survival of cancer cells through the secretion of anti-apoptotic signals, rather than eliminating them.These complex interrelations between cells are typical of predator-prey systems in ecology and can be modelled using similar approaches in which the possible final states (attractors) of the system define which populations will dominate [19]. In any given environment, the cellular processes that determine a cell's phenotype consist in a cascade of interactions, which can be represented as a regulatory network, in which nodes represent proteins, enzymes, chemokines, etc., while the connections represent the type (activation or inhibition) and direction of interactions of different types (transcriptional and post-translational activations).Network modelling has found numerous applications in studying the structure and dynamic behaviour of different biological systems in response to environmental stimuli and internal perturbations [20][21][22][23].Several computational models of different pathways involved in the inflammatory immune response have been previously published, such as: continuous, logical and multi-scale models of T cell differentiation [24][25][26], logical models of macrophage differentiation in pro-and anti-inflammatory conditions [27], multi-scale models of innate immune response in tumoral conditions [28], etc. Macrophages are extremely plastic cell types, whose phenotypes can easily switch depending on conditions.Since the specific macrophage state that protects cancer cells from undergoing apoptosis is fundamental in the development of resistance to treatments in CLL and solid tumors, we turned to study it as a polarization state.An important computational model of macrophage polarization was able to detect 3 different M2 subgroups of macrophages, as a result of various combinations of proand anti-inflammatory extra-cellular signals [27], using exclusively literature-based knowledge of the intra-cellular regulatory interactions and pathways involved in the polarization process.In a more recent work, Ramirez et al. [29] used temporal expression profiles of in vitro macrophage cytokines to infer logical models of macrophage polarization (M1 and 3 subcategories of M2: M2a, M2b and M2c) in the presence of different stimuli.Nevertheless, many important questions remain to be explored regarding the polarization states, especially in a tumor setting.More specifically, it is important to identify the pathways involved in TAM formation and to understand to what extent the macrophage plasticity facilitates this process inside a tumor.On the other hand, despite the wealth of quantitative information from bulk and single-cell sequencing datasets, the inference of regulatory networks based on experimental data remains a difficult challenge, with most approaches proposing a combination of both literature-and data-driven methods [29][30][31][32]. In Chronic Lymphocytic Leukemia (CLL), a B-cell malignancy in which patients accumulate large quantities of malignant CLL cells in their lymph nodes, an interesting ecology of cancer cells and immune cells is established.CLL cells are able to educate surrounding monocytes, through direct contact and cytokine signals, turning them into TAMs, which in this disease are referred to as Nurse Like Cells (NLCs) [33].NLCs are derived from CD14 + monocytes and are characterised by a distinct set of antigens (CD14lo, CD68hi, CD11b, CD163hi) [34,35].Moreover, NLCs express stromal-derived-factor-1alpha, a chemokine which promotes chemotaxis and activates mitogen activated protein kinases, ultimately leading to more aggressive cancers and better survival of these cells in vitro.Through direct contact, the NLCs are able to protect the cancer CLL cells from apoptotic signals, and stimulate environment mediated drug resistance.Interactions between NLCs and CLL cells appear to be mediated by the B cell receptor, which, when stimulated, activates production of CCL3/4, initiating the recruitment of other cells, including CD4 + T cells and more NLCs.Another pathway that has been associated with NLCs and TAMs more in general is that of CSF-1 (MCSF).Patients with high expression of this factor usually show faster CLL progression and this gene was implicated in the production of NLCs.Also the more M1-or M2-like profile of NLCs in specific patients correlates with active and controlled disease, respectively.Analyses of the transcriptomic profile of NLCs suggest their high similarity to the macrophage M2 profile described in solid tumors, which makes studying the formation of NLCs all the more relevant in the quest of controlling TAMs in other malignancies. The main characteristics of these 3 types of macrophages are given in Table 1.Considering the close phenotypic similarity between M2 and NLC/TAM macrophages, we consider that the NLC/TAM components should include the M2 ones.Here we indicate in blue the components that have been used in our model and in bold the ones that were taken as signature components for each phenotype.A schematic diagram of M1 and M2 (with 4 subcategories) macrophages can be found in [2], whereas a short description of the profiles for the main macrophage phenotypes is given in Appendix A. More detailed explanations of the mechanisms, pathways and components involved in the polarization process can be found in the cited papers and the references therein. NLC formation can be studied through an in vitro system in which co-cultures of monocytes and patient-derived CLL cells can be established to produce NLCs in absence of any other cell type.This system is particularly suited to mathematical modelling, as experimental conditions are well controlled and the cell types present are limited to monocytes/macrophages and cancer cells, without the confounding effects of other immune or healthy cells. Boolean models are discrete dynamical models, in which each component (gene, transcription factor, chemokine, cytokine, receptor, etc.) is associated with a discrete (binary) variable, representing its concentration, activity or expression.Despite the complex processes relating the transcription of a gene into an mRNA and its subsequent translation into a protein with possibly post-translational modifications, in this paper we consider a single node for gene, mRNA and protein, such that a link between two transcription factors signifies that one of them affects transcription of the gene coding for the other.The future states of each component are determined by the current states of its regulators, as given by a Boolean function that represents the regulatory relationships between the components according to the logic operators AND, OR and NOT.The state of the system at each time point is given by a binary vector, in which each element represents the state of the corresponding component (ON/OFF) [24,26].Starting from an initial state, as time passes the system will follow a trajectory of states reaching one of many attractors that can be a single stable state (fixed point) or a set of recurrent states (limit cycle).Attractors usually represent specific phenotypes, such as cellular differentiated states, cell cycle states, etc.Despite their coarse-grained description, Boolean models have been successfully used to capture real-world biological features such as, for example, the mechanisms of cell fate decision [45], hierarchical differentiation of myeloid progenitors [46], dynamical modelling of oncogenic signalling [47], among many other applications [48][49][50].One of their main advantages is the simplicity of performing in-silico experiments simulating a variety of mutant and knockout conditions, and the possibility of obtaining qualitative or semi-quantitative results without requiring experimentally derived parameter values, as needed by differential equations.Starting from a pathway diagram describing a biological process, and adding logic rules, Boolean models allow us to model the process, uncover the main regulators, and run simulations. Understanding the mechanisms of TAM formation is of particular interest because of their pro-tumoral activity which hampers T cell cytotoxic activity.In this study, we therefore follow two lines of enquiry: (i) we use a literature and data-driven approach based on an in vitro model of NLC formation to reconstruct a macrophage polarization regulatory network, (ii) we implement a Boolean model of monocyte differentiation into NLC simulating these in vitro cultures. ",
"section_name": "Introduction",
"section_num": "1."
},
{
"section_content": "In this section we describe a model of differentiation of monocytes into macrophages of different polarization states, including the Nurse Like Cell state which is produced in in vitro experiments of monocyte co-culture with cancer cells.We describe how previous models of macrophage polarization were extended to account for this new cell type and compare results of our simulations with existing experimental evidence of the effect of treatments and mutants on the polarization spectrum.Section 2.1 will describe the reconstruction of the regulatory network underlying macrophage polarization while Section 2.2 will explain how Boolean rules were applied to this regulatory network and the dynamics of the resulting model. ",
"section_name": "Results",
"section_num": "2."
},
{
"section_content": "To reconstruct the gene regulatory network (GRN) governing the formation of NLCs, we started from a previous macrophage polarization GRN [27] and extended it in order to include specific extracellular signals found in the Chronic Lymphocytic Leukaemia (CLL) context and other intra-cellular components involved in NLC formation.The network extension was based on extensive literature review and transcription factor (TF) activities estimation for each phenotype. Starting from the model in [27], we further added some nodes based on literature (signals: M-CSF, HMGB1; receptors: M-CSFR, RAGE; intracellular components: IRF5, EGF, TNFα, TGFβ, HIF1α, Table S1a) which describe the mechanisms involved in the establishment of the interaction between NLC and CLL, such as receptor-ligand interaction (M-CSF -M-CSFR, HMGB1 -RAGE [37]) that are frequently present in tumoral environments.We then used public transcriptomics data for monocytes and M1, M2 and NLCs to calculate the TF activities in each phenotype.In these experiments, monocytes were either not induced, induced to mature to macrophages with M-CSF and further activated with Interferon gamma and LPS (M1) or IL-4 (M2) [51,52].The complete analysis of TF estimation on microarray gene expression datasets was performed using the Dorothea R package and Viper [53] and ISMARA [54] (see Methods, Section 5.3).The list of most active TFs for each phenotype (M1, M2 and NLC) from both methods can be accessed in Tables S2a-d and S3a-d. For NLCs, we identified specific TFs using a set of 19 microarray expression profiles we have generated using an in vitro co-culture experiment in which NLCs are differentiated from monocytes upon contact with B cells from a CLL patient [55].Interestingly, we could find a few TFs with higher activities in NLC than in M1 and M2 (Tables S2a,S2d, S3a and S3d).Of this set, we were particularly interested in HIF1α and IRF5, which we added to our model (Table S1a).HIF1α is known to be linked to the pro-tumoral activity of NLC through the HIF1α-dependent activation of CXCL12, an important cytokine secreted by NLCs [52,56].We thus considered this TF among the key regulators for the NLC phenotype.On the other hand, although showing a higher estimated activity in NLC compared to M1 and M2, IRF5 is known to lead to M1-polarization by IL10 repression and TNFα up-regulation [2,57], which is why it was included as a key component in the model but not in any signature (see Section 4 section for further details). The reconstructed regulatory network of macrophage polarization is given in Figure 1.It contains 10 extracellular signals, 30 intra-cellular components, most of them being TFs and interleukins, and 3 outputs, which are used as readouts, namely M1 polarization, M2 polarization and NLC.Pathway enrichment analysis [58] showed that most of the components are involved in the JAK-STAT signalling pathway, pathways related to cancer, Th17 cell differentiation, cytokine receptor interaction and other inflammatory conditions.Having obtained a regulatory network that has the potential to describe the formation of NLCs, we turned to literature to assign to each network node a Boolean rule for its activation. ",
"section_name": "Reconstruction of the Regulatory Network Leading to NLC Formation",
"section_num": "2.1."
},
{
"section_content": "To understand the temporal behaviour of the regulatory system in Figure 1, we model its dynamics by a system of Boolean functions, each of which describes the regulation of the expression of each component.Starting from the Boolean model in [27], we wrote the Boolean functions for the additional nodes based on a supervised literature-based method, using the expression profiles of the macrophage phenotypes taken from the literature (in which most markers of the different macrophage types are protein surface markers).It is important to note that while the NOT operator can be inferred from the regulatory interactions, the AND and OR operators require deeper knowledge of dependent or independent action of regulators on a specific target.We performed a significant number of simulations to explore a wide range of combinations of AND and OR operators and to select the operators' combination that would identify distinct macrophage phenotypes in the attractor space.The Boolean functions for each intracellular component are given in Table 2.The numerical simulations were performed considering all the possible initial intracellular conditions and combinations of stimuli, while applying the synchronous updating method to calculate the system's attractors (Section 5.1).The simulation results show that the system reaches 1384 fixed point attractors, while other cyclic attractors of length 2 and 3 were also present.For our scope, in the following paragraphs we focus only on the fixed point attractors.It is important to note that fixed point attractors are time invariant, i.e., the number of fixed points is not affected from the updating method chosen, while the number of cyclic attractors and their characterisitcs (period, basin of attraction) depend on the updating method (Section 5.1).Here and throughout the paper, we will refer to an attractor as the binarized expression profile which we assign to a polarization state (or a phenotype).To attribute the attractors to certain phenotype categories, we removed all the input nodes (extracellular signals) from the attractors, thus reducing the attractors' space to 214 fixed points. ",
"section_name": "A Boolean Model of Macrophage Polarization",
"section_num": "2.2."
},
{
"section_content": "The large attractors' space raises the challenge of interpreting its biological meaning.To categorize the attractors in specific polarization states, two different methods were used: (1) a supervised literature-based method using the expression profiles of the macrophage phenotypes taken from the literature, and (2) an unsupervised method grouping attractors based on their similarity and then applying clustering algorithms to assign them to specific phenotypes. ",
"section_name": "Phenotype Identification through Interpretation of the Attractors",
"section_num": "2.3."
},
{
"section_content": "To identify the main phenotypes detected by the model, we categorized all the attractors according to the expression profiles of M1, M2 and NLC known from the literature (Table 1 and Appendix A) and the results obtained from TF estimation and gene expression profiles (Table S2c It is important to note that the M1, M2 and NLC categories were considered to be mutually exclusive; therefore the rest of the attractors were categorized as M0, a separate category that includes all the attractors exhibiting characteristics of both M1 and M2 phenotypes, after removing the non-biological states (such as the all-zero attractor and an attractor with all the components in the 0 state except for RAGE = 1).To better characterize the M0 category, we removed the non-biological attractors and re-evaluated the averaged expression profile of the remaining attractors (Figure 2b).As can be seen, this category exhibits neither full M1 nor M2 characteristics, or both (high levels of EGF, IL10R, IL10, IL12, IL1β, IL1R, SOCS1, STAT1) and we believe it corresponds to a non-polarized macrophage, although more detailed characterization might be necessary [66].Despite the existing overlap between markers of M1, M2 and NLCs, we decided to concentrate on the attractors that would have activation of markers of a specific cell type and no activation of others.Clearly other attractors exist that display a mixed phenotype but we decided to classify them separately.Interestingly, we found that most of the attractors fall into the M2 (≈67.3%)category, followed by the M1 (≈4.7%) category and NLC (≈2%) subset (Figure 2).The similarities between attractors falling in each category were estimated by calculating the Jaccard-Needham distances (dist_values ∈ [0, 0.5]).Considering the low values of binary distances between attractors in each category, we then calculated the average attractor states (Figure 2b-e).Importantly, we observe that these averaged attractors largely correspond to the expected expression profiles for M1, M2 and NLC defined above.A principal component analysis shows the main identified clusters of attractors corresponding to each phenotype (Figure 3).From the plot, we can easily observe that NLC attractors are not well separated from M0, which can be explained considering that a large number of attractors in our M0 category have profiles that are intermediate between M1 and M2 and NLCs are also thought to have an intermediate profile.A deeper analysis on identifying the M2 subcategories (M2a, M2b, M2c and M2d) can be found in Text S1, Figures S1 andS2. ",
"section_name": "Interpreting Attractors based on a Supervised Method",
"section_num": "2.3.1."
},
{
"section_content": "Alongside with the supervised method, we also performed unsupervised clustering on the attractor space, in order to investigate whether the main phenotypes we expect in this system can be recovered in an unbiased way just exploring the structure of the attractors' space.We hypothesise that the attractors corresponding to the same phenotype category will be characterized by a small binary distance and consequently will fall into the same cluster.To this end, we first estimated the similarity among the attractors by calculating the Jaccard-Needham distance [67].We then applied hierarchical density-based clustering on the Jaccard-Needham distances (Figure 4) to identify the main attractor clusters.As can be seen from the heatmap, 5 main clusters are detected: one of them (Cluster 4) corresponds to the zero-attractors (attractor 1: all the components in OFF state, attractor 2: all the components in OFF state, except for expr RAGE = 1) and it was not considered for further analysis.A closer look at the averages of the attractors falling in each cluster highlights the detected expression profiles (Figure 4b-e).Based on the averaged expression profiles of attractors in each cluster, we observe a clear representation of M1, M2 and NLC phenotypes, respectively Cluster 5 → M1: IL-12, IL-1R, NF-κB, STAT1, TNFα highly expressed, Cluster 2 → M2: IL-10, IL-10R, JMJD3, KLF4, IRF4, PPARγ and STAT6 highly expressed, and Cluster 3 → NLC: EGF, HIF1α, RAGE, TGFβ and IL-10 highly expressed.Considering the high expression of both M1, M2 and NLC components, we attribute Cluster 1 to M0. ",
"section_name": "Interpreting Attractors Based on an Unsupervised Method",
"section_num": "2.3.2."
},
{
"section_content": "While choosing between supervised and unsupervised methods, one must consider some advantages and disadvantages.Supervised approaches can ensure a specific match between the observed attractors and prior biological knowledge of each phenotype, which can be an issue when the attractors can correspond to uncharacterised biological states and can be limited to the use of existing knowledge.On the other hand, unsupervised methods offer the simplicity of detecting the different state categories in a more unbiased way and possibly to identify unknown intermediate phenotypes in the macrophage polarization spectrum. For a more quantitative comparison between the supervised and the unsupervised methods, we calculated the Pearson correlation coefficient between the averaged expression profiles obtained from each phenotype and each cluster (Figure 5).Our results show the accuracy of the unsupervised method in capturing the M1 (corr_coe f f = 0.92), M2 (corr_coe f f = 1) and NLC (corr_coe f f = 0.91) phenotypes, while the M0 category matches best with Cluster 1 with corr_coe f f = 0.97, not corresponding to any phenotype. ",
"section_name": "Robustness of Attractor Interpretation Independent of Annotation Method",
"section_num": "2.3.3."
},
{
"section_content": "To validate the model, we performed several simulations mimicking specific environmental conditions consisting of M1, M2 or NLC signals only.Previous wet-lab experiments have shown that in co-cultures of monocytes and CLL cells, the CLL signal will elicit the differentiation of monocytes into NLCs.We studied the attractor space in the presence of only CLL signals (M-CSF and HMGB1) while considering all the possible combinations of intra-cellular signals.We then hypothesised that the presence of only a specific phenotype signal inducer (M1, M2 or NLC) would shift the macrophages polarization towards the corresponding phenotype and performed different simulations setting the signals favouring a certain phenotype to the ON state.Indeed, our simulations showed that the presence of specific signals (grouped as M1, M2 and NLC signals) would activate certain pathways that subsequently lead to the corresponding polarization state.Table 3 recapitulates the simulations performed by selecting only specific stimuli, the observed attractors' categories, the expression profiles of each polarization state and the network representation of active/inactive nodes/edges under these conditions.Interestingly, we observed that while the presence of M1 and M2 signals leads to the activation of their corresponding phenotypes, NLC signals activate both M2 and NLC polarization states, which reinforces the shared pro-tumoral activity of both phenotypes in the TME.Additionally, several experimental studies on the effects of mutants and knock-outs on macrophage polarization states have been previously published [62,[68][69][70].Here, we performed simulations of knock-outs and constitutive expressions, as summarized in Table 4. Analysing the attractors' space, we observed a complete loss of M2 phenotype in STAT6 -/-, IRF4-JMJD3 axis KO and a significant decrease of M2 attractors in PPARγ -/-and IL-4Rα -/-, a complete loss of M1 phenotype in IRF5 -/-and STAT5 -/-, and a significant decrease in M1 attractors in STAT1 -/-. Finally, we performed some exploration of our model simulating the knock-out of STAT3 -/-, in which we observed a complete loss of the NLC phenotype with an increase of M1 attractors, and knock-out of EGF -/-, which we predict will also lead to complete loss of the NLC attractors, whereas its constitutive activation (EGF = 1) is predicted to completely eliminate M1 attractors.Interestingly, the simulated knock-down of STAT1 -/-is also predicted to lead to a significant loss of M1.These results show that our model recapitulates the experimental observations in mutant conditions, as well as polarization outputs in the presence of different extracellular signals.They can therefore be used to make predictions that still require experimental validation. ",
"section_name": "Model Validation through In Silico Perturbations",
"section_num": "3."
},
{
"section_content": "The results reviewed in the previous sections highlight the various ways in which network-based dynamic models can be exploited to recapitulate the known characteristics of biological systems, as well as to predict new behaviours in specific conditions.Particularly, despite their limitations to a qualitative description, Boolean models yield a comprehensive picture of a system's dynamics, including all the attractors of the system and the effects of mutants.Here, our main focus lies in identifying the mechanisms that trigger the formation of NLCs in Chronic Lymphocytic Leukaemia, a macrophage polarization state distinct from the ones that can be obtained with monocyte in vitro differentiation.Despite a large body of work on macrophage polarization, the phenotypic profile and formation of tumor associated macrophages have not been fully elucidated yet, due to the difficulty of isolating these cells from tumors.For this reason, we extend a previously published Boolean model of macrophage polarization [27], by including specific nodes (genes, transcription factors and receptors) that characterise the NLC profile.We then apply Boolean rules to the regulatory network to study the system's asymptotic behaviour, when starting from all the possible initial conditions.The main macrophage polarization states (phenotypes) were matched to the attractors first by applying constraints on the value of specific network components (literature-based constraints) and subsequently using unsupervised clustering of the attractors according to their (binary) similarities.Importantly, the model results show that the attractor categories obtained by both supervised and unsupervised methods, qualitatively match the M1, M2 and NLC profiles, while highlighting specific characteristics of NLCs that distinguish them from M2 macrophages.In addition, the unsupervised method, although less accurate than the supervised approach in characterizing the phenotypes, was shown to correctly separate the phenotypic profiles in the absence of any constraint or previous knowledge.Clustering of attractors with more powerful techniques [76,77] would make the unsupervised method suitable especially in Boolean modelling of large networks for which prior biological knowledge is not available. The ultimate test of the model presented would be to compare our in-silico signatures for the different attractors with experimental data measuring the state of each of our model components, possibly through transcriptomic or proteomic characterization of each cell type.However, the multiple levels at which the state of a component can be experimentally determined (gene expression, protein level, protein activation state) reduce our expectations for finding a clear match.Even for the well-characterised biological processes of macrophage polarization, all experimentally derived readouts of the different phenotypes come from the detection of proteins on cell membranes, leaving gaps in our understanding and justifying the need for data-driven approaches.The use of expression data to increase the model accuracy and predictive ability is becoming more and more frequent in the modelling environment and has led to the reconstruction of powerful computational methods to incorporate a wide spectrum of experimental data into the modelling language.However, in this regard, considerable inconsistencies between the literature and experimental data (like TF activity estimation or expression levels in our work) across different methods emphasize the importance of mixed approaches in modelling, especially when aiming for regulatory network inference.For example, it is widely known that IRF5 is involved in macrophage polarization toward a pro-inflammatory M1 state and IRF5 KO is associated with a reduction in expression of pro-inflammatory genes such as iNOS and TNFα, and an increase in genes associated with alternatively activated macrophages, with a loss of the M1 phenotype [70,73,78], in accordance with our KO simulations (Table 4).On the other hand, from our TF activity estimation, we observe a higher IRF5 activity in NLC compared to M1 macrophages.Further work will be devoted to investigating this inconsistency. Here we have shown that we could construct a model of NLC formation starting from information in the literature, a pre-existing model of macrophage polarization and our own transcriptomics data for NLC produced in vitro through differentiation of monocytes in contact with cancer cells.Looking at public transcriptomics and proteomics datasets we were surprised to see relatively little overlap between the well-accepted biomarkers for M1 and M2 and the genes and proteins that are highly expressed in each specific cell type.This could be explained by the important regulatory mechanisms that lead from expression of a gene to the appearance of a protein on the cell membrane (which could be typically detected by FACS), on which most cell type descriptions in immunology are based.Nevertheless, we think that the integration of data in such a model would greatly improve both the usefulness of this model and our understanding of the different phenotypes and we will consider this approach in further work [29,79,80].For example, Ramirez et al. [29] employed transcriptomic time courses to arrive at Boolean models of macrophages in different polarization states, showcasing the potential of integrating experimental data in our mostly literature-based approach. On the other hand, one of the limitations of our model is the choice of a synchronous updating scheme, which prevents us from analysing the cyclic attractors which are known to change depending on the choice of update rules.We think that more biological knowledge about the processes described by this model will be needed to design a better and more appropriate update scheme to then explore the attractor space more deeply. We are also aware that NLCs could represent an artificial phenotype which we observe in the specific in vitro conditions of our experiment and that in patients there could be oscillations spanning all the macrophage phenotypes mentioned.However, since M1 and M2 can clearly be related to the presence of specific stimulatory signals, we think the presence of cancer cells should lead to at least a predominance (over cells in a population or over time) of the NLC state.NLCs have a very specific behaviour in CLL, sending anti-apoptotic signals to the cancer cells, with a very important clinical impact.In the future, we plan to consider more advanced updating schemes that will allow us to explore complex attractors (as mentioned above) which are almost certainly also relevant and could better reflect the nature of NLCs. Taken together, our model can describe macrophage polarization in different environments and mutant conditions.The inflammatory and cancer environments are characterized by a complex combination of stimuli, which drive the polarization process of monocytes towards specific macrophage phenotypes.In our network, we include the most significant pro-and anti-inflammatory signals, as well as important cytokines that are involved in NLC polarization, such as CSF-1 (M-CSF in our model) and HMGB1.Despite the specific characteristics of the tumor micro-environments in solid cancers compared to the in vitro model considered here, we believe that common polarization pathways are also involved in the formation of tumor associated macrophages (TAMs) in solid tumors, which have so far been modeled with a stronger emphasis on the inter-cellular aspects than on the molecular details [81][82][83].Further work will be needed to establish whether our model can be useful more generally in different cellular environments. Despite the limitations of our model and of the in vitro system described, we can allow ourselves to hypothesize about the possible relevance of this model in explaining the mechanistic aspects of formation of TAMs in solid tumors and to suggest potential strategies for hindering their formation.With the advent of immunotherapy, the low response to immune checkpoint blockers that target CD8 + T cells for reactivation in a large proportion of patients remains a serious issue.TAMs are thought to play an important role in this, with the observation that a high macrophage infiltration in tumors in specific indications or patients is often predictive of weaker CD8 infiltration and lower response to current immunotherapies.In particular, over 20% of lung cancer patients carry EGFR mutations that produce a constitutive activation of the EGF receptor [84].We predict an effect of EGF knock-outs and constitutive activations on the proportion of attractors corresponding to M1 and NLC (see Table 4).Patients carrying EGFR mutations show a reduced response to immunotherapy [85], suggesting that further exploration of EGF's role in the formation of TAM might be of clinical relevance.Overall, we hope that our model will encourage new empirical investigations on the complex nature of cell-cell interactions in the TME and the role of TAMs in cancer prognosis and treatment. ",
"section_name": "Discussion",
"section_num": "4."
},
{
"section_content": "",
"section_name": "Methods",
"section_num": "5."
},
{
"section_content": "Here we describe how we implemented a Boolean model starting from the regulatory network described above. In the Boolean model each component (gene/mRNA, protein, chemokine) is associated with a discrete (binary) variable, representing its concentration, activity or expression [86][87][88].Time is considered to be implicit and the future states of each component were determined by the current states of their regulators, given by a Boolean function of m i = 1, 2, . . ., N regulators of component X i .Each Boolean function represents the regulatory relationships between the components and is expressed via Boolean operators AND, OR and NOT.The state of the system at each time point is given by a binary vector, whose ith element represents the state of the component X i [24,26].The set of all possible states and their transitions can be represented by a state transition graph, in which the nodes are the system's states (represented as binary vectors) and the directed edges are the transitions between them.The exponential function between the number of components and the state space size makes the graphical representation possible for only small networks.In Boolean models time is discrete and implicit: starting from an initial state, the system will follow a trajectory of states and, because of the finite state space, it reaches an attractor (stable states or limit cycles).To evaluate the state of each node at each timestep, two main updating methods have been proposed [22,89]: • synchronous updating method: at each time step, all the nodes are updated simultaneously, assuming that all the interactions in the system require the same time to occur.Importantly, the state space is characterized by non-overlapping basins of attractions.• asynchronous updating method: at each time step, the updated nodes are chosen randomly (General Asynchronous, Random Asynchronous) or according to their characteristic updating time, while the system's state will be characterized by overlapping basins of attractions. It is important to note that fixed point attractors are time invariant, i.e., do not depend on the updating method.Choosing the synchronous update method we obtain all transitions between them and consider the final attractors.Our network is composed of N = 40 components (excluding the 3 output nodes which are added for clarity in Figure 1) and has 2 40 possible states.The model was implemented using the BoolNet [69] R package [90]. ",
"section_name": "Boolean Model Implementation",
"section_num": "5.1."
},
{
"section_content": "In this section we give the mathematical background of the similarity measures used for estimating the similarity between different attractors, which we then clustered according to these similarity indexes.Here we hypothesise that the attractors representing a specific phenotype will be characterized by a small distance index (i.e., higher similarity). Given Ω a space of binary N-dimensional vectors Z defined as we define Z = 1 -Z to be the complement of the binary vector Z.For each set of binary vectors Z 1 , Z 2 ∈ Ω let S ij be the number of occurrences of matches, with i ∈ Z 1 and j ∈ Z 2 being in the corresponding positions.In this way S 11 (Z 1 , Z Based on S ij , different measures exist, to calculate the similarity/dissimilarity between two binary vectors [67].For our purpose, we calculated the Jaccard-Needham measures, defined as follows: ",
"section_name": "Calculating the Attractor Similarity Matrix",
"section_num": "5.2."
},
{
"section_content": "Since gene expression of a TF is not representative of its activity level and many of the nodes in our network are TFs, we sought to estimate activity of these components using bioinformatic methods that consider the expression of a TF's targets taken from a dataset in combination with a regulatory network database.We extracted gene expression data from different public sources.Microarray data used in this publication were downloaded from the NCBI repository Gene Expression Omnibus (GEO) database.M1 and M2 Macrophages microarray data accession number is GSE5099 [51,52].Our previously published NLC microarray dataset can be found under accession number GSE87813 and was processed as described in [55].Raw microarray datasets were then normalized using the RMA (Robust Multi-arrays Average) normalization method and batch corrected.Transcription factors activities were estimated using the Dorothea R package and ISMARA.Dorothea is a TF-regulon interaction database giving each interaction a confidence level.Here, levels of confidence of interactions from A to E were taken into account.The VIPER algorithm was used to estimate TF activities based on Dorothea interactions and our expression data [53].ISMARA is a web-based tool to identify the key TFs and miRNAs driving expression/chromatin changes and to predict activities of the regulators across the samples, their genome-wide targets, enriched gene categories among the targets, and direct interactions between the regulators [54].For both methods, the comparison between TF activities across the phoneotypes (M1, M2, and NLC) is performed using the rank method.The complete analysis of TF estimation from both packages, as well as TF activity comparison between phenotypes, can be accessed in Material, Tables S2a-d and S3a-d. ",
"section_name": "Calculating the Transcription Factor Activities",
"section_num": "5.3."
},
{
"section_content": "We present a Boolean model of macrophage polarization in the presence of cancer cells.We showcase two alternative ways to annotate the attractors into previously identified macrophage types, which include M1, M2, and NLC, the specific TAMs encountered in CLL.We validate our model by simulating knockouts that have been experimentally performed in mouse models and make new predictions regarding the importance of STAT and EGF in regulating the production of TAMs, with possible implications for their control in solid tumors.S6: Comparison between gene expression and TF activity estimated with Dorothea/VIPER and ISMARA packages for 19 NLC microRNA samples.It can be seen that the two packages give significantly different results on TF activity, and also considerable difference with gene expression analysis.All distributions (gene expression, TF activities) were independently normalized between 0 and 1 from the results obtained for the NLC phenotype only by subtracting the minimum value min(x) and dividing by distribution range max(x) -min(x).Even if a direct comparison between ISMARA and Dorothea/VIPER TF activity estimated values is not permitted, a lack of correlation is clearly observable.Figure S7: Heatmap of gene expression of 5 monocyte samples, 3 M1 and M2 samples and 19 NLC samples.Table S1a: Macrophage regulatory network extension: added nodes.Table S1b: The main similarities and differences from macrophage regulatory network extension.Table S1c: Boolean rules for the added nodes in the macrophage polarization network.Table S2a: dorothea_results.tsv.Table S2b: Differential activity (Dorothea, M1 > M2 and NLC).Table S2c: Differential activity (Dorothea, M2 > M1 and NLC).Table S2d: Differential activity (Dorothea, NLC > M1 and M2).Table S3a: ismara_results.tsv.Table S3b: Differential activity ranking (ISMARA, M1 > M2 and NLC).Table S3c: Differential activity ranking (ISMARA, M2 > M1 and NLC).Table S3d: Differential activity ranking (ISMARA, NLC > M1 and M2).cytokines including tumor necrosis factor α (TNFα), IL-12, IL-6, IL-1β, IL-18 and IFNα/β [1,36,37].The M1 macrophages metabolism rely on oxidative glycolysis [91] and intrinsically their polarization is linked with activation of STAT1, IRF5 and NF-κB [92].M1-like macrophages are linked in fighting bacterial infections and intracellular pathogens.Additionally they show potent anti-tumoral activity which manifests mainly through: (i) release of large amount of nitric oxide (NO), which in turn is able to kill the cancer cells as a result of DNA damage, disruption of mitochondrial activity and limitation of iron availability, and (ii) presentation of tumor antigens to CD4 + Th1 cells and driving the activity of cytotoxic CD8 + T cells at the tumor site [38]. Appendix A.2. M2 Pathway M2-like macrophages include a wide variety of phenotypes involved in resolving of the inflammation.The M2 activation can be induced by stimulation with IL-4, IL-13, immune complexes and IL-10.The anti-inflammatory and regenerative activity of M2 macrophages come from abundant release of IL-10, TGF-β, VEGF and EGF [1,38].M2 macrophages depend strongly on oxidative phosphorylation [91] and the main TFs driving their polarization-state are: STAT6, PPARγ/δ, IRF4, JMJD3 [92].Depending on the anti-inflammatory processes M2-like macrophages are involved in, they manifest diverse phenotypes including: M2a-Th2 responses and killing and encapsulation of parasites, M2b-immunoregulation, M2c-matrix deposition and tissue remodeling [1][2][3]. Tumor-associated macrophages belong to the group of cells that arise upon the contact with cancer cells and tumor microenvironment (TME).They can show characteristics of both M1 and M2 state, nevertheless upon prolonged presence in the TME the M2 characteristic becomes prevalent.TAMs influence the properties and dynamics of TME, although the precise factors that promote TAM activation have yet to be elucidated, as each TME is characterized by unique physical and chemical conditions [13,38].However, certain common features may be identified.For example, CSF1, IL-10 and TGF-β released from tumor cells and Treg cells, are powerful promoters of TAM polarization, which in turn support tumor progression by various mechanisms, such as: (i) secretion of soluble immunosuppressive agents (IL-10, TGF-β, IL-1β), (ii) expression of Immune Checkpoint Inhibitors (PD-L1, B7-H4), and (iii) high levels of hypoxia-inducible factor 1 and 2 (HIF1, HIF2) which leads to expression of genes associated with pro-tumoral activity [12,13,38,93]. In the context of Chronic Lymphocytic Leukaemia (CLL) it has been proposed that Nurse-like cells (NLC), which are specific form of TAMs identified in this malignancy, are polarized in response to CSF-1 and HMGB1 proteins released by CLL cancer cells.In turn NLCs can stimulate and protect CLL cells by antigen presentation which stimulates BCR signaling, and also by both direct contact through membrane proteins and release of soluble factors including: [37] • membrane proteins: CD2 (interacts with LFA-3 expressed on CLL cells [55]), CD31 (ligand of CD38 expressed on CLL cells), BAFF, APRIL (both BAFF and APRIL can be also released as soluble factors) [ ",
"section_name": "Conclusions",
"section_num": "6."
}
] |
[
{
"section_content": "The authors thank Alexis Coullomb, Ting Xie, Julien Pernet, Maria Fernanda Senosain Ortega and Julie Bordenave for advising and critical reading of the manuscript. ",
"section_name": "Acknowledgments:",
"section_num": null
},
{
"section_content": "Funding: This research was funded by INSERM, the Fondation Toulouse Cancer Santé and Pierre Fabre Research Institute, as part of the Chair of Bioinformatics in Oncology of the CRCT. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "Author Contributions: Conceptualization, M.M. and V.P.; methodology, M.M. and V.P.; software, M.M., F.R. and M.M.-M.; resources, M.M., N.V., F.R., M.M.-M., M.D., M.P., J.-J.F., L.Y. and V.P.; validation, M.M., F.R., M.M.-M., N.V. and V.P.; data curation, F.R., M.M.-M.; original draft preparation, M.M., V.P. and N.V.; review and editing, M.M., F.R., M.M.-M., N.V., V.P., M.D. and L.Y.All authors have read and agreed to the published version of the manuscript. The authors declare no conflict 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. The M1-like pro-inflammatory polarization state is applied to pro-inflammatory macrophages and can be obtained upon stimulation of those cells with IFNγ or LPS which cause release of Th1-inducing Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. ",
"section_name": "Conflicts of Interest:",
"section_num": null
},
{
"section_content": "Author Contributions: Conceptualization, M.M. and V.P.; methodology, M.M. and V.P.; software, M.M., F.R. and M.M.-M.; resources, M.M., N.V., F.R., M.M.-M., M.D., M.P., J.-J.F., L.Y. and V.P.; validation, M.M., F.R., M.M.-M., N.V. and V.P.; data curation, F.R., M.M.-M.; original draft preparation, M.M., V.P. and N.V.; review and editing, M.M., F.R., M.M.-M., N.V., V.P., M.D. and L.Y.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.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
},
{
"section_content": "The M1-like pro-inflammatory polarization state is applied to pro-inflammatory macrophages and can be obtained upon stimulation of those cells with IFNγ or LPS which cause release of Th1-inducing Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. ",
"section_name": "Appendix A.1. M1 Pathway",
"section_num": null
}
] |
10.1186/s42047-019-0052-9
|
Literature review of Notch melanoma receptors
|
<jats:title>Abstract</jats:title><jats:p>Despite the immunotherapeutics and target therapy agents, the survival of patients with advanced melanoma is still low. Notch signaling is able to regulate many aspects of melanomagenesis. Comparative analyses of common melanocytic nevi, dysplastic nevi and melanomas demonstrated increased expression of Notch1, Notch2 and their ligands, indicating that a positive regulation of these components may be related to the progression of melanoma. Some strategies such as gamma-secretase inhibitors (GSI) have been explored in patients with refractory metastatic disease or locally advanced disease of solid tumors. Two major classes of Notch inhibitors are currently in clinical development: GSI and monoclonal antibodies against Notch receptors or their ligands. Inhibition of Notch by GSI has been shown to decrease melanoma growth. GSI RO4929097 co-administered with cisplatin, vinblastine and temozolomide promotes greater elimination of tumor cells. The Notch pathway needs to be explored in the treatment of melanoma.</jats:p>
|
[
{
"section_content": "After gastrulation, the neural crest is first induced at the edge of the neural plate and in the non-neural ectoderm.Neural crest cells are initially multipotent but gradually become restricted to the potential line of development, which is determined by where they migrate and settle.Those that migrate to the epidermis and dermis, are subjected to the specification of lineages to form precursors of melanocytes -melanoblasts.Neural crest cells can originate several types of differentiated cells and tissues, including sensory neurons and glial cells, melanocytes, cartilage and craniofacial bone and smooth muscle.Melanocytic nevus is a benign accumulation of melanocytes that may be considered the first lesion in progression, evolving to melanocytic hyperplasia, dysplasia and melanoma.Dysplastic lesions may progress to melanoma in situ, invasive melanoma and then metastatic melanoma.This multi-stage tumor process results from the accumulation of genetic alterations that comprise genomic instability, activation of oncogenes such as BRAF and RAS, as well as inactivation of tumor suppressor genes (Bevona et al. 2003;Gandini et al. 2005;Hussein 2004;Naeyaert and Brochez 2003;Cichorek et al. 2013). Analysis of the Cancer Genome Atlas and other genomic data made it possible to identify the most frequent mutations in melanoma and other cancers.BRAFV600E is the most prevalent mutation in melanoma, detected in 52% of cases, followed by mutations in the RAS and neurofibromin 1 (NF1) family in approximately 30 and 14% of cases, respectively.These conductive mutations are almost always mutually exclusive, making it possible to classify melanoma cases in distinct genomic subtypes: BRAF, RAS, NF1 and Triple-WT, the latter being defined by the absence of BRAF, RAS and NF1 mutations.Other common changes in melanoma include mutations in TP53, CDKN2A and TSGs homologous to phosphatase and tensin (PTEN) and in the TERT promoter (Akbani et al. 2015;Curtin et al. 2005). Recent discoveries in cell signaling mechanisms have provided a better understanding of the biology underlying the progression of melanoma, and these advances have been explored to provide targeted drugs and novel therapeutic approaches.Among the genetic events underlying the development of melanoma, a variety of mutations in the components of cell signaling pathways was characterized, notably in the MAPK kinase pathway.Many of these alterations impair the functions of receptors on the plasma membrane and the aligned signaling cascades (Ghosh and Chin 2009;Gray-Schopfer et al. 2007;Dantonio et al. 2018). ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "Cutaneous melanoma originates from normal melanocytes or their precursors and among skin cancers is responsible for the highest number of deaths.In humans, melanocytes can be detected in the epidermis around the 50th day of intrauterine life.Embryonally, these cells migrate from the neural crest to the basal layer of the epidermis along the dorsolateral pathway.It has been suggested that the process of melanocyte development is often used by tumor cells to promote their own progression.The worldwide incidence of melanoma has been increasing in recent decades.The US National Cancer Institute reports that the rate of new cases of cutaneous melanomas has increased by an average of 1.5% per year over the past 10 years (Yoshida et al. 2001;Liu et al. 2014;Shain and Bastian 2016). Familial melanoma accounts for 8-12% of melanoma cases, with the cyclin-dependent kinase inhibitor 2A (CDKN2A) and CDK4 being the major susceptibility genes involved in this context.Germline mutations in CDKN2A are responsible for the loss of two tumor suppressor proteins, p16INK4a and p14ARF, both encoded by the CDKN2A gene through alternative splicing while germline mutations in the CDK4 oncogene make a complex constitutively active between CDK4 and cyclin D1, which promotes abnormal cell proliferation (Thompson et al. 2009;Potrony et al. 2015;Aoude et al. 2015;Bertolotto 2013;Zuo et al. 1996). Cutaneous melanoma affects young individuals (world average age 64 years) and presents aggressive behavior, with high lethality.Despite the breakthrough in advanced disease therapy, with the emergence of immunotherapeutics (anti-PD1 and anti-CTLA4) and target therapy agents (anti-BRAF and anti-MEK), survival of cutaneous melanoma patients remains directly related to the stage of the disease at diagnosis and depends mainly on the depth of the skin lesion as measured by the Breslow index.Thus, early diagnosis is of fundamental importance as well as the emergence of new therapeutic agents (National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology 2018; Mozūraitienė et al. 2015). In mammals, four Notch receptors have been identified (Notch1-4) and are considered transmembrane type I proteins with the function of activation of transcription factors involved in embryonic development and whose signaling pathway is responsible for the control of epidermal differentiation and proliferation in adult life.Ligands on the surface of the neighboring cells (Delta1, Delta3, Delta4, Jagged1 and Jagged2) activate the Notch receptor inducing its intramembrane cleavage by gamma secretase complex which results in the release of the intracellular Notch domain in its active form with consequent activation of a series of genes involved in differentiation of progenitor cells during development (Massi et al. 2006;Panelos and Massi 2009). The Notch signaling pathway contributes to the regulation of the melanocyte lineage.During development, melanocyte precursors, melanoblasts, emerge from the neural crest and migrate to the epidermis through newly formed hair follicles.In the hairless regions, melanoblasts remain immature and located in the basement membrane of the epidermis where they are differentiated for mature melanocytes by stimulating adjacent keratinocytes or other signals from the microenvironment.Recent studies indicate that the Notch signaling pathway is important for the maintenance of melanoblasts, for the appropriate localization of melanoblasts in the epidermal basement membrane, and for the induction of melanocyte differentiation in the follicular matrix.The transformation of normal melanocytes into melanoma cells is a multi-step process.MAPK is the major route involved in the known melanomagene.Activation of Notch signaling may contribute to melanoma progression, although it is not known which cells serve as emitting cells.Notch signaling is mediated by direct interactions between membrane-bound Notch receptors (Notch1-4) in recipient cells and Notch ligands (Delta or Jagged type) in the emitting cells (Massi et al. 2006;Panelos and Massi 2009;Golan et al. 2015;Guruharsha et al. 2012). The Notch signaling cascade is initiated after binding of the membrane bound linker (Jagged 1,2 and DSL 1,3,4) to Notch receptors.After binding of the ligand, two sequential proteolytic events occur to release the active intracellular Notch (NIC).The first cleavage is mediated by a metalloprotease, TNF-a converting enzyme (TACE).Final cleavage is mediated by a γ-secretase complex.Subsequently, the NIC translocates to the nucleus and functions as a transcription factor to influence gene expression, including HES, Hey and other targets (Liu et al. 2014). Some studies suggest that the Notch signaling pathway is pro-tumorigenic in melanomas.Notch1 expression levels are elevated in 50-60% of melanomas and 65% in melanoma cell lines, while in normal melanocytes and nevi are low or undetectable.In addition to promoting growth and survival of melanoma cells, Notch1 is also involved with a more aggressive phenotype.This phenotype has been associated with the activation of the MAPK and Akt pathways (Golan et al. 2015;Bedogni 2014;Kumano et al. 2007;Okuyama et al. 2008b). It has now been demonstrated that in melanomas there is a greater expression of microRNAs (miR-146a) due to the activation of MAPK pathway (resulting from BRAF or mutant NRAS) and increased transcription factor MYC. Overexpression of miR-146a leads to the suppression of NUMB, which is a Notch-regulated protein. The silencing of NUMB by overexpression of miR-146a releases the Notch signaling pathway leading to the growth of tumor cells in melanoma (Golan et al. 2015;Garraway 2014). In addition to the activation of the Notch signaling pathway involved in cell proliferation, it is also responsible for acquiring the vertical invasion capability of cutaneous melanomas (Golan et al. 2015). The Notch signaling pathway in melanomas is activated directly by the contact between melanocytes and differentiated keratinocytes (keratinocytes from the most superficial layers of the epidermis) through their ligands (Delta-like or Jagged).After cell-cell (differentiated melanoma-keratinocyte) activation, the intracellular Notch domain (NICD) is cleaved by gamma-secretase complex and translocated to the nucleus where it binds to a transcriptional regulatory protein, the Jkappa protein (RBPJK) and its cofactor MITF.The RBPJK / MITF complex is responsible for the inhibition of the miR-222/221 micro RNA promoter, thus increasing the expression of miR-222/221, considered the trigger for vertical invasion in melanomas (Golan et al. 2015). The Notch pathway is a highly conserved cell pathway, present in most multicellular organisms, which plays an important role in the determination, proliferation, differentiation and survival of the cell.The Notch signaling cascade modulates a wide range of cellular processes, including regulation of cell cycle blockade, apoptosis, survival, differentiation and maintenance of stem cells, as well as the response to hypoxia.Consequently, the aberrant function of Notch is involved in a number of human diseases, including developmental disorders, neurodegenerative diseases and cancer (e.g., T cell leukemia, multiple sclerosis, lymphoma and melanoma) (Ranganathan et al. 2011;Gustafsson et al. 2005;Osawa and Fisher 2008;Joutel and Tournier-Lasserve 1998;Müller 2012). The Notch family of proteins is composed of cell surface receptors that transduce signals interacting with the Delta-like (DLL) and Jagged (JAG) transmembrane ligands in neighboring cells.In mammals, a total of four Notch receptors (Notch1-4) and five ligands were characterized (DLL1, 3, 4 and JAG1 and 2).Notch receptors are composed of two main domains: an extracellular domain (NECD) and an intracellular domain (NICD), interconnected by a transmembrane protein.The NICD has two structures at the C-terminus that are responsible for nuclear input (Kopan 2012;Takebe et al. 2014;Kojika and Griffin 2001;LaVoie and Selkoe 2003). All Notch receptors are synthesized as full-length proteins that undergo successive proteolytic cleavages, culminating in the release of NICD, which will act on the nucleus.Notch ligands located in the neighboring cells have a Delta / Serrate / Lag-2 (DSL) domain that mediates binding-receptor binding and thus promotes the activation of Notch signaling.The mechanism for triggering Notch canonical signaling involves proteolytic cleavages at three Notch sites: S1, S2 and S3.The first cleavage (S1) is mediated by furin type convertases, which occurs in the trans-Golgi network during the Notch secretion process.This cleavage generates two subunits (NECD and NICD) interconnected by the transmembrane protein.These processed subunits are then transported to the plasma membrane, where they associate as non-covalently linked heterodimers to form the functional Notch receptor.Upon binding of the linker to the receptor, the latter undergoes the second cleavage (S2) which is catalyzed by a member of the disintegrin A family and metalloproteases (ADAM17 or ADAM10), also known as tumor necrosis factor (TACE) converting enzyme.Cleavage of S2 promotes the dissociation of the membrane-bound intracellular Notch domain from the extracellular domain.This intracellular domain is a constitutive substrate for the final cleavage of S3, culminating in the release of active NICD, which is directed to the nucleus by its NLS.S3 is regulated by a protease complex of the presenilin-dependent γsecretase, which consists of an integral membrane protein complex.After translocation to the nucleus, the NICD binds to CSL (transcriptional repressor RBPJk / CSL), a transcriptional repressor that acts as a DNA binding adapter and helps recruit the adapter protein (MAML), which in turn evokes the transcriptional coactivator p300 and other components of the transcription machinery (Liu et al. 2014;Kopan 2012;Takebe et al. 2014;Kojika and Griffin 2001;LaVoie and Selkoe 2003;Baron 2003;Pinnix and Herlyn 2007;Okuyama et al. 2008a;Nam et al. 2003). The transcriptional activation complex, composed of CSL, NICD, MAML and p300, regulates transcription of genes downstream, such as HES1 and HEY.The Hess family of repressors acts downstream of the Notch signaling pathway, antagonizing the expression of a variety of transcription factors, such as Ascl1, Atoh1 and Neurog3, whose function is to keep the cells in an undifferentiated state.Hes repressor genes, such as HES1, play key roles in maintaining progenitor cells in an undifferentiated state.The concomitant existence of transcriptional activators and repressors downstream of the Notch signaling cascade plays a crucial role in the various outcomes of this pathway (Osawa and Fisher 2008;Pinnix and Herlyn 2007;Tao et al. 2010;Kageyama et al. 2007). Several studies have also demonstrated the existence of noncanonical activation of Notch signaling in several cell types.At least three types of non-canonical activation of Notch have been described, which may or may not depend on: (i) ligand interaction, (ii) γ-secretase activity and (iii) the action of RBPJk / CSL complexes.Both non-canonical cascades independent of RBPJ and Hes have important functions downstream of Notch signaling, although the exact molecular events mediating these sub-pathways are not fully understood (Osawa and Fisher 2008;Veeraraghavalu et al. 2005;Hodkinson et al. 2007;Ayaz and Osborne 2014). Notch signaling may also interact with other pathways, such as PI3K, mTORC2, Wnt, NF-κB, YY1 or HIF-1α, at cytoplasmic and / or nuclear levels.Direct interactions of NICD with IKKα in the NF-κB pathway or LEF1 in the Wnt pathway were reported, and that Notch can activate the integrin via Ras, irrespective of RBPJ binding.Although many normal cellular processes (homeostatic regulation of melanocytes) require canonical signaling of Notch, many pathological conditions, including cancer and immune system activation, are associated with noncanonical Notch signals.However, these noncanonical cascades still require a more detailed understanding (Tao et al. 2010;Ayaz and Osborne 2014;Hirobe 2005). In healthy skin, Notch signaling is expressed in all layers of the epidermis and is critical for the differentiation of keratinocytes and affects a wide range of cellular activities including cell cycle arrest, apoptosis, and survival.An example of the versatile function of the Notch pathway is related to epidermal development, where Notch signaling is able to stimulate the differentiation of granular cells into spinous cells and, at the same time, prevent their premature differentiation (Okuyama et al. 2008a;Hirobe 2005). This versatility of functions can be explained by the concomitant existence of a transcriptional activator and a repressor downstream of Notch.Notch signaling, via Hes1 transcription factor, maintains the survival of melanoblast and melanocytic stem cells, preventing the onset of apoptosis (Osawa and Fisher 2008;Moriyama et al. 2006). Notch signaling is able to regulate many aspects of melanomagenesis.Comparative analyses of common melanocytic nevi, dysplastic nevi and melanomas demonstrated an increased expression of Notch1, Notch2 and its ligands, indicating that a positive regulation of these components may be related to the progression of melanoma (Massi et al. 2006). An essential role for the Notch pathway has been validated in the development of melanoblast as well as the progression of melanoma.Notch1 is considered a primary tumorigenic factor in melanoma.Positive regulation of Notch1 and its target genes occurs in metastatic melanoma.Transcriptional targets of Notch signaling that are responsible for angiogenesis, proliferation, metastasis and cell survival in tumor cells include Hes, Hey, Cyclin D1, NF-κB, STAT3 and p21 (Schouwey et al. 2007;Ma et al. 2014;Balint et al. 2005). The Notch pathway may offer a therapeutic possibility for the treatment of melanoma.Considering the effects of Notch overexpression on the development of melanoma, the manipulation of this pathway seems to have great potential therapeutic value.Several strategies to inhibit the Notch pathway have been used against melanoma and other cancers.Recent evidence has also suggested that Notch signaling is one of the most important cell pathways in drug-resistant tumor cells.In fact, negative regulation of the Notch pathway may induce drug sensitivity, leading to increased inhibition of the growth, invasion, and metastasis of cancer cells.For example, the use of RNA directed to Notch2 has shown potential to reduce tumor invasion and the growth of uveal melanoma, a common intraocular malignancy in adults (Liu et al. 2014;Purow 2009;Wang et al. 1806;Asnaghi et al. 2012). Increasing evidence indicates that Notch signaling contributes to physiological processes, including development, differentiation and tumorigenesis, either as a promoter or tumor suppressor, depending on the cellular context, level of expression and interaction with other signaling systems.There is also evidence that signaling through Notch receptors regulates cell proliferation and cell survival in various types of cancer, including melanoma.Tumor progression / melanoma metastasis are complicated processes that require multiple cellular events, including cell proliferation, survival, migration and invasion.Notch signaling appears to be a promising system for novel therapeutic targets for the treatment of melanoma and perhaps for the prevention of melanoma metastases (Müller 2012). Increased knowledge of the molecular basis of melanoma has transformed the treatment of these patients.Advances in immunobiology brought a class of immunotherapeutic agents that provided more durable control of patients with metastatic melanoma.For tumors that do not respond to immunotherapeutics but have specific mutations as in BRAFV600E (occurring in approximately 50% of cutaneous melanoma cases), target therapies with MAPK pharmacological inhibitors (RAF and MEK effectors) have shown benefit, even though not all of melanomas with the mutations respond to the MAPK pharmacological inhibitors (Tolcher et al. 2012). Despite the benefits, approximately 40% of melanomas do not respond to inhibitors of the MAPK pathway nor to target therapy agents.Tumors develop resistance and patients have recurrences (Wolchok et al. 2017).Some strategies such as gamma-secretase inhibitors have been explored in phase I and phase II studies in patients with metastatic refractory disease or locally advanced disease of solid tumors.It has been investigated whether the addition of gamma-secretase inhibitors to RAF and MEK inhibitors could bring benefit in the treatment of patients with advanced melanoma (Krepler et al. 2016;Tolcher et al. 2012). Two major classes of Notch inhibitors are currently in clinical development: γ-secretase inhibitors (GSIs) and monoclonal antibodies (mAbs) against Notch receptors or their ligands.Inhibition of Notch by the y-secretase inhibitor has been shown to decrease melanoma growth.A phase I clinical trial for GSI RO4929097 has shown encouraging evidence of antitumor activity, revealing itself as a selective inhibitor of small molecules of ysecretase with antitumor activity.Suppression of Notch-2 activation is critical for honokiol to inhibit melanoma cells (Kaushik et al. 2015). Antitumor activity by GSIs and mAbs administered as a single agent in the early stages of clinical trials has been observed in advanced or metastatic thyroid cancer, non-small cell lung cancer, intracranial tumors, sarcoma or desmoid tumors, colorectal cancer with neuroendocrine features, melanoma and ovarian cancer.Several adverse events, particularly gastrointestinal toxicities, have been observed and mitigation strategies are being developed following the testing of multiple GSIs and Notchdirected mAbs (Takebe et al. 2014). Combined chemotherapy with GSIs for recurrent and advanced stage melanoma were evaluated in Phase I and II clinical trials.GSI RO4929097 co-administered with cisplatin, vinblastine and temozolomide promotes a greater elimination of tumor cells (Nickoloff et al. 2005;Briot and Iruela-Arispe 2015;Espinoza and Miele 2013).There is evidence that the Notch pathway needs to be explored because of its potential to become useful in the treatment of melanoma. ",
"section_name": "Main text",
"section_num": null
},
{
"section_content": "Considering the effects of Notch overexpression on the development of melanoma, the manipulation of this pathway seems to have a great potential therapeutic value.Several strategies to inhibit the Notch pathway have been used against melanoma and other cancers.Despite the benefits, approximately 40% of melanomas do not respond to immunotherapy (anti-CTLA4, anti-PD1 and anti PDL1) nor to target therapy agents (anti-BRAF and anti-MEK).It has been investigated whether the addition of gamma-secretase inhibitors to BRAF and MEK inhibitors could bring benefit in the treatment of patients with advanced melanoma. Two major classes of Notch inhibitors are currently in clinical development: γ-secretase inhibitors (GSIs) and monoclonal antibodies (mAbs) against Notch receptors or their ligands.GSI co-administered with cisplatin, vinblastine and temozolomide promotes a greater elimination of tumor cells (Nickoloff et al. 2005;Briot and Iruela-Arispe 2015;Espinoza and Miele 2013).Notch pathway needs to be explored because of its potential to become useful in the treatment of melanoma. ",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "None ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "CDKN2A: Cyclin-dependent kinase inhibitor 2A; DLL: Delta-like; DSL: Delta / Serrate / Lag-2; GSI: Gamma-secretase inihibitors; JAG: Jagged; mAbs: Monoclonal antibodies; NECD: Nocth extracellular domain; NF1: Neurofibromin 1; NIC: Active intracellular Notch; NICD: Intracellular Notch domain; PTEN: Phosphatase and tensin; TACE: TNF-a converting enzyme Authors' contributions RSOF conceived of the study and participated in its design and coordination.ALS provided the search of the main subject in databases Medline and LILACS.FMP drafted the manuscript.EO and RM contributed with pathology aspects of the study.ACB and LMF participated in the sequence alignment and final revision.All authors read and approved the final manuscript. 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": "Abbreviations",
"section_num": null
},
{
"section_content": "CDKN2A: Cyclin-dependent kinase inhibitor 2A; DLL: Delta-like; DSL: Delta / Serrate / Lag-2; GSI: Gamma-secretase inihibitors; JAG: Jagged; mAbs: Monoclonal antibodies; NECD: Nocth extracellular domain; NF1: Neurofibromin 1; NIC: Active intracellular Notch; NICD: Intracellular Notch domain; PTEN: Phosphatase and tensin; TACE: TNF-a converting enzyme Authors' contributions RSOF conceived of the study and participated in its design and coordination.ALS provided the search of the main subject in databases Medline and LILACS.FMP drafted the manuscript.EO and RM contributed with pathology aspects of the study.ACB and LMF participated in the sequence alignment and final revision.All authors read and approved the final manuscript. ",
"section_name": "Abbreviations",
"section_num": null
},
{
"section_content": "",
"section_name": "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": "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/s13148-016-0274-6
|
Global DNA methylation profiling reveals new insights into epigenetically deregulated protein coding and long noncoding RNAs in CLL
|
Methyl-CpG-binding domain protein enriched genome-wide sequencing (MBD-Seq) is a robust and powerful method for analyzing methylated CpG-rich regions with complete genome-wide coverage. In chronic lymphocytic leukemia (CLL), the role of CpG methylated regions associated with transcribed long noncoding RNAs (lncRNA) and repetitive genomic elements are poorly understood. Based on MBD-Seq, we characterized the global methylation profile of high CpG-rich regions in different CLL prognostic subgroups based on IGHV mutational status.Our study identified 5800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal controls. From cllDMGs, 40 % of hypermethylated and 60 % of hypomethylated genes were mapped to noncoding RNAs. In addition, we found that the major repetitive elements such as short interspersed elements (SINE) and long interspersed elements (LINE) have a high percentage of cllDMRs (differentially methylated regions) in IGHV subgroups compared to normal controls. Finally, two novel lncRNAs (hypermethylated CRNDE and hypomethylated AC012065.7) were validated in an independent CLL sample cohort (48 samples) compared with 6 normal sorted B cell samples using quantitative pyrosequencing analysis. The methylation levels showed an inverse correlation to gene expression levels analyzed by real-time quantitative PCR. Notably, survival analysis revealed that hypermethylation of CRNDE and hypomethylation of AC012065.7 correlated with an inferior outcome.Thus, our comprehensive methylation analysis by MBD-Seq provided novel hyper and hypomethylated long noncoding RNAs, repetitive elements, along with protein coding genes as potential epigenetic-based CLL-signature genes involved in disease pathogenesis and prognosis.
|
[
{
"section_content": "High-throughput next-generation sequencing techniques, with single base pair resolution have become increasingly feasible, along with the existing genomic and transcriptome sequencing methodologies.These techniques have been successfully used to understand the functional role of DNA methylation in leukemia development and progression, including CLL.Somatic hypermutations of the IGHV gene have been shown to be a strong prognostic marker in CLL, where CLL patients with an unmutated IGHV gene have poor prognosis and shorter survival time compared to IGHV-mutated CLL patients [1,2].Previously, using high-resolution 27K/450K methylation arrays in CLL, we analyzed the global methylation profiles of well-characterized prognostic groups such as IGHVmutated and IGHV-unmutated CLL subsets [3][4][5][6].Our data identified a large number of differentially methylated genes with prognostic implications for the CLL prognostic subgroups, and importantly, we found that the methylation patterns were stable over time and between the compartments [3,5].In addition, using 450K methylation arrays and whole genome bisulphite sequencing (WGBS) techniques, a recent investigation characterized the DNA methylomes of CLL patients and found that differential methylation in the gene body may have functional and clinical implications in leukemogenesis [7,8].The most common methodologies used in all these CLL studies include microarray and sequencing methods which are based on bisulfite conversion of genomic DNA for differentiating 5-methyl cytosine (5mC) from cytosine (C). However, bisulfite conversion-based methodologies have some drawbacks; these methods fail to differentiate between 5mC and other epigenetic modifications such as 5hmC (hydroxyl methyl cytosine) and 5cmC (carboxyl methyl cytosine) [9,10], and they may also not be the best methods for characterizing repeat sequences in the genome.Instead, other techniques like affinity-based enrichment methods such as MBD-Seq or Methylated DNA immunoprecipitation, followed by sequencing (MeDIP-seq) can overcome these drawbacks and provide genome-wide coverage of CpG methylation in a PCRunbiased manner.These immunoprecipitation-based enrichment of CpG methylated DNA methods in CLL provide DNA methylation profiling for both protein coding and noncoding RNAs, as well as repeat regions which have not yet been studied.A recent study showed very good correlation between 450K methylation array and MeDIP-seq on a genome-wide scale.However, MeDIP-Seq allowed wider interrogation of methylated regions in the human genome, including some nonreference sequences that were not included in the array and also the methylation of repetitive elements [11]. Noncoding RNAs (ncRNAs) have been shown to regulate important biological functions such as maintenance of nuclear architecture, X-chromosome inactivation [12], and genomic imprinting [12,13].ncRNAs can be broadly classified into long noncoding RNAs (lncRNAs), micro-RNAs (miRNAs), antisense RNAs, small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs).Like proteins, ncRNA modulate transcription and play regulatory roles in controlling the localization and activity of proteins [14][15][16][17].The precise distribution and temporal expression of ncRNAs in the genome are important for cellular homeostasis.Deregulation of the expression of ncRNAs leads to several disorders including cancer [16,18,19], and recent studies underline the emerging role of ncRNAs as biomarkers in different malignancies [20][21][22].Even though global differential expression patterns of ncRNAs were observed between CLL cells and corresponding healthy controls [23,24], studies on the novel epigenetically deregulated ncRNAs in CLL are limited. In order to investigate CLL-associated differentially methylated genes compared to normal healthy controls, we performed MBD-Seq to ascertain the global distribution of the methylomes between five IGHV-mutated and five IGHV-unmutated CLL patient samples.Additionally, we also compared the methylomes of each subgroup with healthy age-matched controls, against PBMCs and sorted B cells separately.This is the first MBD-Seq-based CLL study, revealing many CLL-specific significantly methylated protein coding genes, noncoding RNAs, and certain repetitive regions with potential prognostic significance. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "Patient samples, ethics, clinical data, cell lines, and cell culture conditions In the present study, a total of 70 CLL patients (35 IGHV-unmutated samples + 35 IGHV-mutated samples) were included.All patients were diagnosed according to recently revised criteria [25] and the tumor samples were collected at the time of diagnosis.The patients in the study were included from different hematology departments in the western part of Sweden after written consent had been obtained.Only CLL peripheral blood mononuclear cells (PBMC) samples with a tumor percentage of leukemic cells ≥70 % were selected in this study.Clinical and molecular data are summarized in Additional file 1A and B. PBMCs from peripheral blood of age-matched normal healthy controls was prepared using the Ficoll extraction method and normal CD+19 positive sorted B cell DNA from eight healthy agematched controls were bought from a company (3H Biomedicum, Uppsala, Sweden).Two CLL cell lines (HG3 [26] and MEC1 [27]) and one Burkitt lymphoma B cell line (RAMOS) [28] were used for DAC treatment experiments.All cell lines were cultured in RPMI 1640 with glutamine (Invitrogen, Carlsbad, USA) supplemented with 10 % fetal bovine serum and 1× penicillin/streptomycin (FBS; Invitrogen, Carlsbad, USA). ",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "DNA and RNA were extracted from CLL PBMC samples using DNA and RNA Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.For total cDNA preparation, reverse transcription (RT) was performed using Superscript III FS synthesis supermix kit (Life technologies, Carlsbad, USA) according to the manufacturer's protocol. ",
"section_name": "DNA and RNA extractions",
"section_num": null
},
{
"section_content": "Purified genomic DNA from CLL patient samples were subjected to sonication using bioruptor (Diagenode, Liege, Belgium) to generate fragment sizes of around 100 to 350 bp.The fragmented DNA was then subjected to MethylMiner TM methylated DNA kit enrichment according to the manufacturer's protocol and the enriched methylated DNA was purified using single fraction extraction with buffer containing 2000 mM NaCl.The eluted DNA was purified and sent for downstream library construction and high-throughput MBD-Seq using the Illumina HIseq2000 platform.The analysis has been done using five IGHV region-mutated and five IGHV region-unmutated patients samples along with normal PBMC and normal B cell as control samples.The raw sequenced reads (FASTQ files) from Illumina for two sorted BCELL, two PBMC, five IGHV-mutated, and five IGHVunmutated samples are deposited on European Nucleotide Archive (ENA) under project ID \"PRJEB12693\" and can be accessed via following link http://www.ebi.ac.uk/ena/ data/view/PRJEB12693.The raw reads from sequencing were cleaned for adaptors using Trimmomatic [28], and bioinformatics analysis has been performed on those clean reads.The hg19/GRCh37 genome version was used to map obtained 49-bp cleaned reads.The alignment was performed using Bowtie v1.0 aligner by allowing up to two mismatches.It is a short-read aligner supports up to length of 50 bp [29].We used an additional parameter -m 6 in Bowtie to reduce the number of multiple aligned reads. ",
"section_name": "Methyl-binding domain sequencing and data preparation",
"section_num": null
},
{
"section_content": "The differentially methylated regions were predicted using MACS v1.4.2 peak caller [30] by not considering duplicate reads at exact location.The analysis has been carried out by assigning four different groups, two prognostic groups (IGHV-mutated and IGHV-unmutated PBMC), and two normal group (sorted B cell and PBMC normal).The normal groups were used as control in MACS and obtained positive peaks (enriched in prognostic groups) were termed as hypermethylated over normal whereas the negative peaks as hypomethylated (enriched in normal sample).This analysis was done using each control groups as different comparison (B cell comparison and PBMC comparison).All obtained CLL cllDMRs presented in Fig. 1c were enriched with a p value < 1e-05 (represents peak score, 50) and the enrichment heatmaps were obtained from a standard R package.The global methylation levels (as shown in Fig. 1d) represents percentage of bases in the genome occupied by enriched regions in normal B cell, PBMC, and IGHV-mutated and IGHV-unmutated CLL samples, and the analysis was done using bedtools-genomecov. ",
"section_name": "Differential methylation and functional significance (association of differentially methylated regions to genes in the genome)",
"section_num": null
},
{
"section_content": "Association of cllDMRs to genes was done using HOMER [31] tool with Ensembl transcript annotation version GRCh37.74 and using default parameters in HOMER.We termed the genes associated with differentially methylated regions as differentially methylated genes (cllDMGs).After association, the genes from each prognostic subgroups (IGHV-mutated and IGHV-unmutated) were compared (for example, in Fig. 2a).There were some genes appeared in both subgroups, and we termed it as common cllDMGs.The genes which only fall in either of one subgroup we termed it as subgroup specific cllDMGs (IGHVmutated specific and IGHV-unmutated specific cllDMGs).These terminologies were used for both hyper and hypomethylated genes (Fig. 2a,b) in all comparisons (B cell and PBMC normal comparisons).The percentage of repeat sequence covered by cllDMRs was obtained using RepeatMasker (http://repeatmasker.org) including all repeat elements as reference.We have used sequence from each peak region predicted by MACS to find sequence similarity (with minimum insertions or deletions) with known repeat elements using Repeatmasker.The clustering of repeat elements in Fig. 5a and Additional file 2: Figure S3A were done by \"euclidean\" as distance metric (complete-linkage clustering) using percentage of bases covered in cllDMRs by different repeat elements obtained. ",
"section_name": "Association of cllDMRs with different genomic regions and analysis of repeat regions",
"section_num": null
},
{
"section_content": "The pathway enrichment analysis and cancer enrichment analysis on cllDMGs was carried out with the help of a command line functional enrichment tool called Gen-eSCF v1.1 (Gene Set Clustering based on Functional annotation) [32,33].We used GeneSCF with parameters, two different database KEGG pathways and NCG (Network of cancer Genes 4.0) [34], Ensembl GRCh37.74 protein coding genes as background genes.The resulted KEGG pathways were filtered with a p value <0.05 with at least 5 % of total genes covered for particular pathway.For cancer enrichment analysis, we used a threshold of at least 5 % of total genes covered for corresponding cancer type.The GeneSCF ranks the pathways and cancer types with p values obtained from Fisher's Exact test using total protein coding genes in the experiment as a background.The Fisher's exact test is carried out based on overlaps between cllDMGs and the genes from corresponding databases (NCG or KEGG).The original list of all significant pathways (as presented in heatmaps from Fig. 2d, Additional file 2: Figure S1D andS2C) and cancer types (as presented in heatmaps from Fig. 2c, Additional file 2: Figure S1C andS2B) was listed as same order in Additional files 3 and 4. ",
"section_name": "Pathway enrichment of cllDMGs and their enrichment in different cancer types",
"section_num": null
},
{
"section_content": "Since the available processed dataset from Ferreira PG et al. [23] used different gene level annotation (GENCODE), we wanted to maintain the same annotation throughout our study (Ensembl).We obtained the raw data of RNA-seq samples for 96 patients (55 IGHV-mutated and 41 IGHVunmutated prognostic groups) along with 9 normal B cell samples (Controlled Access ICGC dataset at the EGA, EGAD00001000258).The obtained samples are from paired-end with 76-bp length reads.The raw reads were subjected to adaptor cleaning using Trimmomatic, and the cleaned reads with 76-bp length was aligned to hg19 genome using a spliced read mapper Tophat v2.0.13 with default parameters.Reads were quantified for Ensembl annotation (GRCh37.74)using featureCounts from Subread packag v1.4.5 [35].The obtained gene expression profile was normalized to reads per kilobase of transcript per million mapped reads (RPKM).The log-fold changes between B cell normal and two CLL groups (IGHV-mutated and IGHV-unmutated) were calculated based on obtained RPKM values.The statistics for differential expression between normal and CLL prognostic groups was obtained using Wilcoxon rank sum test in R package.The methylation patterns from pyrosequencing of CRNDE and AC012065.7 was inversely correlated with the gene expression patterns in RNA-seq dataset (Fig. 4a,b).The p values in this heatmap were presented as Wilcoxon rank sum test. ",
"section_name": "Processing and comparing cllDMGs with RNA sequencing expression data obtained from published CLL data set",
"section_num": null
},
{
"section_content": "The lncRNAs in cllDMGs from B cell and PBMC comparisons were used to extract nearby protein coding genes within 10 kb using bedtools \"-closest.\"Functional and cancer enrichment analysis for obtained nearby protein coding genes were performed by GeneSCF v1.1 using KEGG and NCG as reference database. ",
"section_name": "Nearby protein coding genes analysis",
"section_num": null
},
{
"section_content": "Pyrosequencing was performed as previously described [36], using the Pyromark kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.Pyrosequencing primers were designed using the PyroMark™ (Qiagen, Hilden, Germany) software (FP-5′GGAAAAGG GGAGGTAAAGAGG3′; RP-5′TACCTTTACAAAAATC CTACCAAAATA CTA3′; and sequencing primer-5′ GGTAGTTTAGAAGTTTTTGTTAGTT3′ (280 bp product size) for CRNDE); (FP-5′AGTTTTTGTTTAGATT TTTGGTTGTTAGA3′; RP-5′AAAAA ATATATACAAT TACACCAACTCAC3′; and sequencing primer-5′GTA TTTTGTTGAATTA GAAGGA3′ (222-bp product size) for AC012065.7)and (FP-5′GTTTATAGATATGGTTA GA ATGGG3′; RP-5′TCCCCAATAACTAAAACTACA AACT3′; and sequencing primer-5′ATA TGGTTAGA ATGGGT3′ (236-bp product size) for CLL IGHV-mutated specific SINE-ALU repeat).The analysis was performed using PyroMark™ Q24 advanced pyrosequencer instrument and the CpG site methylation percentage of target regions was calculated using the PyroMark Q24 advanced software.The expression levels of all genes were analyzed with Taqman gene expression assays (Applied Biosystems) (Hs00395639_m1 for CRNDE, custom assay designed primers for AC012065.7 and Hs99999907_m1 for the b2-microglobin gene, which was used as an internal control).Differences in expression were calculated using the ΔΔCt method. ",
"section_name": "Pyrosequencing and real-time quantitative PCR",
"section_num": null
},
{
"section_content": "Correlations between overall survival and methylation or gene expression were calculated using the Kaplan-Meier method and the log-rank test.Differences were considered statistically significant when the p value was <0.05 (Fig. 4c, Additional file 2: Figure S3A). ",
"section_name": "Overall survival analysis for validated genes",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "A brief overview of the work-flow used in this study is shown in Fig. 1a, b, which summarizes both the experimental work-flow and the computational pipeline used to analyze MBD-Seq data generated from CLL patients and normal healthy controls.In this study, the genomic DNA from five IGHV-mutated favorable prognostic and five IGHV-unmutated poor prognostic CLL samples were used, along with CD19+ sorted B cells and total PBMCs as normal controls obtained from two to three different pooled age-matched healthy controls (Fig. 1a). As an initial step of analysis, we extracted the differentially methylated regions, which are specifically hypermethylated or hypomethylated in CLL samples compared to the normal sorted B cell controls.These regions were named CLL-specific differentially methylated regions (cllDMRs) and were defined as enriched regions from IGHVmutated and IGHV-unmutated samples compared to control samples with a p value <0.00001.Both cllDMRs and methylated repeat regions in the genome were fine mapped and then compared between different CLL .The cancer types are assigned and ranked using GeneSCF.The presented enrichment was filtered using a p value <0.01 with at least 5 % of total cancer genes covered by DMGs.d The heatmap shows the KEGG pathways obtained using DMGs from IGHV-mutated and IGHV-unmutated prognostic groups.The pathways were assigned and ranked using GeneSCF.The presented pathways are filtered using a p value <0.01 with at least 5 % of total pathway genes covered by DMGs (see the \"Methods\" section).The left side of the heatmap represents the subgroup specific (IGHV-mutated and IGHV-unmutated) hyper-and hypomethylated associated pathways; and the right side of the heatmap for common DMGs between IGHV-mutated and IGHV-unmutated groups (see the \"Methods\" section) panel), are shown.The genes associated with cllDMRs were termed CLL-associated differentially methylated genes (cllDMGs). ",
"section_name": "Experimental design and mapping of CLL-associated differentially methylated regions",
"section_num": null
},
{
"section_content": "When compared the total percentage of genome covered by MBD-seq samples from CLL prognostic groups and normal healthy controls, we found that IGHV-unmutated samples showed less genome coverage compared to IGHV-mutated samples (Fig. 1d).The genome coverage denotes number of bases in the genome covered by aligned reads from corresponding samples.The overall genome coverage of IGHV-mutated samples were almost in the same range as normal controls such as PBMC and sorted B cells.The coverage levels of the normal PBMC sample were in a higher range compared to the normal sorted B cell sample as shown in the Fig. 1d.Interestingly, even though we found that the overall genome coverage of IGHV-mutated and IGHV-unmutated cllDMRs showed a similar pattern, there is a clear difference in distribution of cllDMRs between the hypermethylated and hypomethylated groups with respect to protein coding genes.According to the cllDMRs distribution data across the genome, hypermethylated cllDMRs are mostly enriched in promoter regions TSS (transcriptional start sites) and TTS (transcriptional termination sites), whereas hypomethylated cllDMRs are enriched in the gene body and intergenic regions (Fig. 1e). The obtained cllDMRs were further associated with different classes of CLL-specific differentially methylated genes (cllDMGs), like protein coding and noncoding genes based on the overlapping genomic locations of cllDMRs as listed in Additional files 5 and 6.Nearly 50 % of cllDMRs, from IGHV-mutated and IGHV-unmutated CLL prognostic groups, map to ncRNAs (lncRNAs, microRNAs, snRNAs, snoRNAs, and pseudogenes) (Fig. 2a, b for B cell control and Additional file 2: Figure S1A andB for PBMC control comparisons).This data is in line with the published RNA-seq CLL study showing that many lncRNAs are differentially expressed in CLL compared to normal healthy controls and that DNA methylation could be one of main reasons behind their differential expression [23]. When enrichment for different cancer types was tested using hypermethylated and hypomethylated cllDMGs from CLL IGHV-mutated and IGHV-unmutated groups, the CLL cancer type was significantly enriched in both B cell and PBMC cllDMGs (Fig. 2c, B cell comparison; Additional file 2: Figure S1C, PBMC comparison; and Additional file 2: Figure S2B, common genes between B cell and PBMC comparison).In the B cell cllDMGs, along with CLL, lung and pancreas cancer types were also found to be significantly differentially methylated as shown in Fig. 2c.The detailed list of cancer type enrichments with the corresponding list of cllDMGs is shown in Additional files 3 and 4, and see the \"Methods\" section for enrichment analysis.On the other hand, hypermethylated and hypomethylated common cllDMGs (hypermethylated or hypomethylated in both prognostic subgroups) were highly enriched in several lymphomas, including CLL and other solid tumors like prostate, colorectal, and breast cancer.More importantly, the overlapped common cllDMGs (851 hypermethylated and 2061 hypomethylated) between B cell and PBMC control comparisons (Additional file 2: Figure S2A) showed significant enrichments in cancer types related to leukemia (Additional file 2: Figure S2B).These results show that most of the commonly deregulated cllDMGs are cancer associated genes, and they may have a functional role in CLL pathogenesis. ",
"section_name": "Characterization of cllDMRs across the genome",
"section_num": null
},
{
"section_content": "We next performed a functional analysis to investigate pathways that were potentially deregulated by DNA methylation in CLL.Several novel as well as already implicated pathways in CLL have shown significant enrichments either in IGHV-mutated specific methylated (hypo or hyper) genes or unmutated specific methylated (hypo or hyper) genes or commonly methylated (hypo or hyper) genes between two prognostic groups (Fig. 2d and Additional file 2: Figure S1D).Notably, some important pathways were specifically deregulated in CLL such as ErbB, B cell receptor, PI3K-Akt, Wnt signaling, and MAP Kinase signaling (Fig. 2d and Additional file 2: Figure S1D).The detailed KEGG pathway summary with percentage of genes involved in each pathway along with the p values is listed in Additional file 3 (for B cell and PBMC comparisons) in the same order as in the corresponding heatmaps presented.Interestingly, most of these pathways like the B cell receptor, MAP Kinase, and PI3K-Akt pathways were also significant when analyzed for common cllDMGS from Additional file 2: Figure S2A between B cell and PBMC comparisons (Additional file 2: Figure S2C and Additional file 4). ",
"section_name": "Biological pathways deregulated by DNA methylation in CLL",
"section_num": null
},
{
"section_content": "Even though some of the cllDMGs were commonly hypermethylated or hypomethylated in all CLL patients, many genes showed significant differences in the methylation scores between the two prognostic groups.To further investigate the correlation between DNA methylation and gene expression, we used published RNA-seq data from a CLL cohort comprising 98 patients [23].First, we selected a few commonly methylated cllDMGs in all CLL samples and sub-divided into two groups based on promoter methylation or gene body methylation [7] (Fig. 3a).There were nearly 24 had promoter hypermethylation with lower gene expression and 42 cllDMGs with hypomethylated promoters showing higher gene expression.Consistent with recent analysis, gene body methylation positively correlated with gene expression [7]: 42 cllDMGs had hypermethylated gene body with higher gene expression and lower expression in the case of 84 cllDMGs with hypomethylated gene body.All the selected cllDMGs had greater than 50 peak score (in both IGHV-mutated and IGHVunmutated groups) and with expression of log-fold change >±1 over normal B cell (in both IGHV-mutated and IGHV-unmutated groups) (Fig. 3b,c).Since these selected genes from MBD-seq and RNAseq data sets showed the expected correlation patterns between DNA methylation and gene expression, they serve as a vital resource for uncovering their possible role in CLL prognosis.In order to extend these observations, we selected two lncRNAs to further validate the significance of DNA methylation in gene expression using pyrosequencing and qRT-PCR methods respectively in an independent CLL cohort (the selected genes are highlighted in Fig. 3b).The selection of genes was based on both methylation peak scores and ncRNAs; however, we have excluded genes that were not suitable for designing pyrosequencing primers due to high CpGrich regions.Since the significance of DNA methylation in the differential expression of lncRNAs has not been investigated in CLL, we selected lncRNAs are CRNDE and AC012065.7,which were hyper-and hypomethylated on promoter regions respectively (Fig. 3b).The methylation levels were validated using pyrosequencing in 48 CLL patients and 6 sorted B cell healthy controls, and as expected, we found that this data was in line with the MBD-seq data, where a higher percentage of methylation for all CLL samples was observed compared to normal samples (Fig. 4a).qRT-PCR analysis revealed that the DNA methylation levels of CRNDE and AC012065.7 showed an inverse correlation to gene expression levels in the same sample cohort (Fig. 4b, right panel), suggesting that these two cllDMRs may play a functional role in regulating the gene expression of cllDMGs.More importantly, our qRT-PCR data further corroborates with published independent RNA-seq data (with total 98 samples) (Fig. 4b, left panel). We also analyzed the prognostic value of these two lncRNAs using Kaplan-Meier analysis.Both CRNDE and AC012065.7 lncRNAs showed a significant correlation between overall survival and DNA methylation in CLL patients (Fig. 4c).Higher methylation levels of the CRNDE promoter and lower methylation levels of the AC012065.7 promoter correlated with poor overall survival (Fig. 4c). Importantly, in order to explore the causal role of DNA hypermethylation in regulating CRNDE expression, we treated three different leukemic cell lines (HG3, MEC1, and RAMOS) with increasing concentrations of the methyl inhibitor (5′-Aza-2′-deoxycytidine, also known as DAC).As shown in Fig. 4d, a corresponding increase of CRNDE expression was demonstrated for all DAC treated samples compared to untreated samples in all the three cell lines, supporting that this gene is deregulated mainly due to hypermethylation on promoter region (Fig. 4d). ",
"section_name": "Correlation between candidate cllDMGs methylation and expression",
"section_num": null
},
{
"section_content": "We next investigated the expression correlation between lncRNAs from cllDMGs and nearby protein coding genes using RNA-seq datasets from 96 CLL patient cohorts.LncRNA AC012065.7,which is promoter hypomethylated with higher expression in CLL compared to normal (upregulated), showed positive expression correlation with nearby protein coding gene GDF7 (Fig. 4e).GDF7 is known to play an important role in growth, repair, and embryonic development, and its polymorphism leads to adenocarcinoma.Similarly, CRNDE also showed positive expression correlation with its neighboring protein coding gene IRX5 (Fig. 4e).The gene IRX5 has been shown to be involved in apoptosis and cell cycle regulation in prostate cancer cells [37].Since the nearby protein coding genes of two selected lncRNAs has cancer related functions, we were interested in understanding the functional significance of nearby protein coding genes which are 10 kb proximity to all lncRNAs from cllDMGs (Additional file 7).The functional and cancer enrichment analysis revealed that cancer terms such as leukemia and lymphoma and KEGG pathways such as Wnt signaling (nearby genes from B cell and PBMC (See figure on previous page.)Fig. 3 Regulation of cllDMGs by the distribution of methylation on gene structure and the gene expression patterns associated with methylation.a Table showing the selection of candidate genes depending on correlation between location of methylation on gene structure (promoter or gene body methylation in MBD-seq) and their pattern of gene expression (up or downregulated in RNA-seq, log2fold-change).The \"selected number of genes\" in green represents the candidate genes (cllDMGs) considered for further investigation.The two selected genes (CRNDE and AC012065.7)for further investigation from two categories were highlighted in bar graphs (b and c) with a rectangle.b The top and bottom bar graphs represent the list of selected cllDMGs from promoter-hypermethylated-downregulated and promoter-hypomethylated-upregulated patterns, respectively.c The top and bottom bar graphs show the list of selected cllDMGs from gene body-hypermethylated-upregulated and gene body-hypomethylated-downregulated patterns, respectively a e d b Fig. 4 Validation of differential methylation and expression levels in CLL cohorts.a Boxplots on top shows the difference in distribution and level of methylation between IGHV-mutated, IGHV-unmutated, and sorted B cells for two selected genes (CRNDE and AC012065.7)obtained using pyrosequencing.b The boxplots shows the difference in gene expression levels between IGHV-mutated, IGHV-unmutated, and sorted B cells for same genes obtained using published RNA sequencing dataset (Ferreira PG et al.) and quantitative RT-PCR.The heatmap below each boxplot shows the significance level (p value) of the corresponding gene over B cell (IGHV-M, IGHV-mutated, and IGHV-UM, IGHV-unmutated).c Kaplan-Meier plots showing the clinical significance of all the validated genes based on high and low methylation levels.The high and low levels were calculated using upper and lower quartile based method for all the genes in total 44 CLL patient samples.d Gene expression levels of CRNDE using increasing concentrations of DAC treatment in different leukemic cell lines.e The illustrations (left panel) represents the protein coding genes IRX5 and GDF7 within 10-kb proximity of selected lncRNAs CRNDE and AC012065.Global methylation analysis of repetitive elements in the IGHV-mutated and IGHV-unmutated CLL samples Global hypomethylation in cancer cells can be largely attributed to reduced methylation of repetitive elements in the genome [38][39][40].To this end, we investigated the percentage of repeat sequence covered by the cllDMRs and found that SINE-ALUs, satellites, simple repeats, and LINEs which were enriched in significantly hypomethylated regions from CLL samples compared to the normal controls (Fig. 5a).Moreover, the IGHV-unmutated poor prognostic CLL samples showed less enrichment of these repeat elements in hypomethylated regions compared to the IGHV-mutated CLL samples, further supporting the hypothesis that SINE and satellite repeats are hypermethylated in healthy normal controls and IGHV-mutated samples.We also investigated if repetitive elements were enriched in hypermethylated regions of CLL samples compared to normal and found enrichment of SINE-ALUs in all CLL samples.Interestingly, these SINE-ALUs were more in hypermethylated regions from the IGHVunmutated group compared to the IGHV-mutated group (Fig. 5a and Additional file 2: Figure S3A).We selected one methylated SINE-ALU repeat sequence which was more enriched in the IGHV-mutated prognostic group compared to the IGHV-unmutated prognostic group in our analysis and validated it using pyrosequencing (Fig. 5b). ",
"section_name": "LncRNA from cllDMGs show significant expression correlation with neighboring protein coding genes",
"section_num": null
},
{
"section_content": "Using a high-throughput affinity-based methylated DNAenrichment technique, for the first time, we analyzed global methylomes of two different CLL prognostic groups to identify DNA methylation based protein coding, lncRNA, and repeat RNA signatures by comparing to two different kinds of healthy normal controls; both sorted B cells and PMBCs.More than half of the cllDMGs were revealed to be common between B cell and PMBC comparisons, sharing many significant common biological pathways.These observations suggest that the common differentially methylated genes from these two comparisons could be a huge resource for investigating epigenetic-based signatures for on 450K methylation array data, CLL has been stratified into three groups with similarity to naïve B cells, memory B cells, and an intermediate group [7,41].However it is also true that many known prognostic markers and candidate genes were identified in CLL by comparing with normal sorted B cells, such as ZAP70 [42], BCL2 [43], and ANGPT2 [36].Moreover, the exact corresponding healthy control for CLL is not clear as the cell of origin of this B cell leukemia is still under debate.Therefore, based on many recent published global methylation studies in CLL [3,5,8,44] where they used normal B cell as controls, we also used sorted B cells and PBMCs in our study to identify CLL-associated hyper/hypo methylated genes.Several lines of evidence suggest that CLL genomes are hypomethylated compared to normal sorted B cells [45][46][47][48].Since MBD-Seq investigates methylation on the genome-scale in an unbiased manner, it is an ideal methodology to address the global CpG methylation levels in CLL subsets in relation to sorted B cells and PBMCs.We found that IGHV-unmutated samples exhibit significant overall global hypomethylation compared to IGHVmutated samples, whose methylation levels were comparable to normal healthy controls, which is consistent with their favorable clinical prognosis.Interestingly, when we compared the distribution of cllDMRs across the genome, we observed that hypermethylated cllDMRs were enriched in promoter regions, whereas hypomethylated cllDMRs were significantly enriched over gene body and intergenic regions further supporting the above statement. Moreover, this is the first detailed study where both CLL-associated hypermethylated and hypomethylated cllDMRs were investigated in unbiased manner across genic, intergenic and repeat regions of the genome.Unlike MeDIP-seq, which enriches regions with relatively lower CpG density, MBD-seq mostly enriches regions with slightly higher CpG density [49].Interestingly, all the cllDMRs showed high GC content (more than 50 to 55 %), which was expected based on above mentioned study (the percentage of GC content and CpG content for all the cllDMRs are mentioned in the Additional files 5 and 6 for both B cell and PBMC comparisons, respectively).In this study, we also investigated the grade of enrichment of the cllDMGs in other cancer types and found that CLL was the top-listed among the leukemias (Fig. 2c).Also, the enrichment of common cllDMGs in other lymphomas and cancers such as non-Hodgkin lymphoma, colorectal, and prostate cancer indicates that these could be signature DMRs for cancers in general, including CLL. Lately, there has been a clear shift in researchers' focus towards lncRNAs and understanding their role in cancer initiation and progression [50,51].However, their relative importance in hematological disorders is still limited.A recent RNA-seq based CLL study identified many differentially expressed lncRNAs as potential biomarkers in CLL pathogenesis [23]; however, the mechanisms underlying their differential expression is still unknown.In the current study, a significant portion (nearly 40 % of hypermethylated and 60 % of hypomethylated) of differentially methylated transcripts were ncRNA, comprising small ncRNAs like microRNAs, snRNAs, snoRNAs and lncRNAs, such as lncRNAs, pseudogenes and antisense transcripts.This large dataset of ncRNAs could also be a resource for further studies, aiming at understanding the functional role of ncRNA in CLL pathogenesis.Towards this end, we validated the differential expression of two lncRNAs (AC012065.7andCRNDE) in an independent cohort.Both these lncRNAs were found to be hyper and hypomethylated using both normal B cell and PBMC comparisons as listed in Additional file 6. Another important aspect of the current study, unlike previously published methylation array studies, is that we have performed extensive correlation studies of cllDMG methylation and gene expression using the published RNA-seq data set from 98 CLL patients [23].We found several protein coding RNAs and lncRNAs showing strong correlation between DNA methylation and gene expression.For example, cllDMGs with hypermethylation of the promoter had lower gene expression levels, whereas cllDMGs with gene body hypomethylation had higher gene expression (Fig. 3).Our qRT-PCR and pyrosequencing has validated the gene expression and DNA methylation levels, respectively, of selected lncRNAs (CRNDE and AC012065.7).Moreover, hypermethylation and hypomethylation of CRNDE and AC012065.7 lncRNAs, respectively, correlated with inferior overall survival, and since there are no other lncRNAs identified in CLL as epigenetic prognostic markers, it would be interesting to further investigate these two lncRNAs for their potential prognostic role in CLL. Finally, we found that several KEGG pathways in CLL, including MAPK, PI3K-AKt, and B cell receptor signaling, were enriched with cllDMGs.Moreover, the analyses of CLL samples in relation to both B cell and PBMC normal controls revealed several common KEGG pathways, and many of these pathways were also listed in a recent RNA-seq study in CLL [23], implying that methylation could be a determining factor in the aberrant regulation of these pathways.Also, some of these pathways, like Notch [48,52,53] and NF-kappa B [53,54] have already been implicated in CLL. Transposable elements such as LINEs and SINEs are enriched with CpG sites and therefore DNA methylation levels of these repeat regions serve as a robust surrogate marker of global DNA methylation [38,40].CpG methylation analysis of repeat sequences is not possible with bisulfite converted microarray-based techniques.Hence, until now, data about the possible relevance of repeat region methylation in CLL has been scarce.We found that many repeat regions like SINE/Alus, LINE/Alus, LTR/ERV, satellites, and simple repeats were significantly hypomethylated in both prognostic CLL subgroups (Fig. 5a).Considering that LINE repeats along with SINE/Alu repeats constitute more than 40 % of the total human genome, it is generalized that hypomethylation of these repeats results in global demethylation [55].On the other hand, we also identified specific SINE/Alu repeats which were significantly hypermethylated in both IGHV-mutated and IGHV-unmutated CLL subgroups against normal B cell controls, indicating a pathogenic function of hypo/hypermethylated specific SINE/Alu repeats in CLL.The exact mechanism by which these repeat regions may increase the risk of cancer is unclear; however, it has been hypothesized that cells with higher methylation levels may have a longer survival, and thus, combined with carcinogen exposure, this methylation pattern may favor clonal expansion of damaged cells [56].We then validated one of the IGHV-mutated specific hypermethylated SINE/Alu repeat region using pyrosequencing in a larger CLL cohort comprising 70 tumor samples.However, we could not validate SINE/ Alu repeat regions that were more hypermethylated in IGHV-unmutated samples due to a high GC content.So, further work is needed to realize their significance as prognostic biomarkers in CLL. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "In summary, for the first time using MBD-Seq, we investigated global CLL-specific methylomes using sorted B cells and PBMCs as controls.We identified several lncRNAs, including CRNDE and AC012065.7,repetitive elements (SINE/Alu), and protein coding RNAs harboring cllDMRs with a potential role in CLL disease pathogenesis and/or prognosis.Also, our data opens up several important CLL-associated pathways for further investigations. ",
"section_name": "Conclusions",
"section_num": null
}
] |
[
{
"section_content": "We thank Dr. Hallgerdur Kristjandottir for her contribution in providing CLL samples and Giti Shah Barkhordar for providing clinical data for CLL patient samples from Sahlgrenska University hospital.\"The computations were performed on resources provided by Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX) high-performance computing (HPC) which is part of Swedish National Infrastructure for Computing (SNIC).\" ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This study was supported by the Swedish Research Council (dnr E 2012/126), the Swedish Cancer Society (CAN 2013/386), Knut and Alice Wallenberg Foundation (KAW) (Dnr KAW 2014.0057),Swedish Foundation for Strategic Research (RB13-0204), Barncancerfonden (PR2014/0147), and \"FoU Västra Götalandsregionen\" (ALFGBG-507731). ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "The data set supporting the results of this article is included within the article (and its additional files). ",
"section_name": "Availability of data and materials",
"section_num": null
},
{
"section_content": "Authors' contributions SS performed the research and analyzed the data.SS, POA, STK, and CK analyzed the data and wrote the paper.MK was the principal investigator and also performed the research, analyzed the data, and wrote the paper.All authors read and approved the final manuscript. POA has consulted for/had an advisory role with Janssen-Cilag Sweden, GSK Sweden, and Gilead Sweden and has served on a speaker's bureau for GSK Sweden.The other authors declare that they have no competing interests or disclosures. Not applicable for this study. The Regional Ethics Review Board, Gothenburg, approved the study.The ethical approval for collecting our CLL samples is from 2007-05-21 with the following registration number: EPN Gbg dnr 239/07.No studies were done involving animals in this study. ",
"section_name": "Additional files",
"section_num": null
},
{
"section_content": "Authors' contributions SS performed the research and analyzed the data.SS, POA, STK, and CK analyzed the data and wrote the paper.MK was the principal investigator and also performed the research, analyzed the data, and wrote the paper.All authors read and approved the final manuscript. ",
"section_name": "Additional files",
"section_num": null
},
{
"section_content": "POA has consulted for/had an advisory role with Janssen-Cilag Sweden, GSK Sweden, and Gilead Sweden and has served on a speaker's bureau for GSK Sweden.The other authors declare that they have no competing interests or disclosures. ",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Not applicable for this study. ",
"section_name": "Consent for publication",
"section_num": null
},
{
"section_content": "The Regional Ethics Review Board, Gothenburg, approved the study.The ethical approval for collecting our CLL samples is from 2007-05-21 with the following registration number: EPN Gbg dnr 239/07.No studies were done involving animals in this study. ",
"section_name": "Ethics approval",
"section_num": null
}
] |
10.1186/s13045-017-0450-y
|
Next-generation sequencing and FISH studies reveal the appearance of gene mutations and chromosomal abnormalities in hematopoietic progenitors in chronic lymphocytic leukemia
|
Chronic lymphocytic leukemia (CLL) is a highly genetically heterogeneous disease. Although CLL has been traditionally considered as a mature B cell leukemia, few independent studies have shown that the genetic alterations may appear in CD34+ hematopoietic progenitors. However, the presence of both chromosomal aberrations and gene mutations in CD34+ cells from the same patients has not been explored.Amplicon-based deep next-generation sequencing (NGS) studies were carried out in magnetically activated-cell-sorting separated CD19+ mature B lymphocytes and CD34+ hematopoietic progenitors (n = 56) to study the mutational status of TP53, NOTCH1, SF3B1, FBXW7, MYD88, and XPO1 genes. In addition, ultra-deep NGS was performed in a subset of seven patients to determine the presence of mutations in flow-sorted CD34+CD19- early hematopoietic progenitors. Fluorescence in situ hybridization (FISH) studies were performed in the CD34+ cells from nine patients of the cohort to examine the presence of cytogenetic abnormalities.NGS studies revealed a total of 28 mutations in 24 CLL patients. Interestingly, 15 of them also showed the same mutations in their corresponding whole population of CD34+ progenitors. The majority of NOTCH1 (7/9) and XPO1 (4/4) mutations presented a similar mutational burden in both cell fractions; by contrast, mutations of TP53 (2/2), FBXW7 (2/2), and SF3B1 (3/4) showed lower mutational allele frequencies, or even none, in the CD34+ cells compared with the CD19+ population. Ultra-deep NGS confirmed the presence of FBXW7, MYD88, NOTCH1, and XPO1 mutations in the subpopulation of CD34+CD19- early hematopoietic progenitors (6/7). Furthermore, FISH studies showed the presence of 11q and 13q deletions (2/2 and 3/5, respectively) in CD34+ progenitors but the absence of IGH cytogenetic alterations (0/2) in the CD34+ cells. Combining all the results from NGS and FISH, a model of the appearance and expansion of genetic alterations in CLL was derived, suggesting that most of the genetic events appear on the hematopoietic progenitors, although these mutations could induce the beginning of tumoral cell expansion at different stage of B cell differentiation.Our study showed the presence of both gene mutations and chromosomal abnormalities in early hematopoietic progenitor cells from CLL patients.
|
[
{
"section_content": "Chronic lymphocytic leukemia (CLL) is characterized by the clonal proliferation and accumulation of neoplastic B lymphocytes in the blood, bone marrow, lymph nodes, and spleen [1,2].Immunophenotype analysis of CLL cells shows expression of CD5 T cell antigen as well as CD19, CD20, and CD23 B cell surface antigens [3].In molecular terms, CLL is defined by the presence of chromosomal abnormalities (11q-, +12, 13q-, 17p-) that play an important role in CLL prognosis [4].The mutational status of the immunoglobulin heavy chain (IGHV) is also considered a prognostic marker in CLL [5,6].Recently, the development of next-generation sequencing (NGS) techniques has enabled mutations to be identified in novel target genes in CLL [7,8], and mutations in some drivers such as NOTCH1, SF3B1, TP53, and MYD88 genes have been shown to have a prognostic impact in CLL patients [9][10][11]. The cellular origin of this disease remains controversial [12][13][14].Recent studies have reported that CLL pathogenesis may start at a previous maturational cell stage, or even in hematopoietic stem cells (HSCs).Fluorescence in situ hybridization (FISH) studies showed that +12 and 13q-abnormalities are present in CD34+CD19 cells, suggesting that these common chromosomal abnormalities could appear in HSCs [15,16].Interestingly, xenotransplantation studies reported that HSCs from CLL patients were able to reproduce the CLL phenotype in murine models [17].In addition, CLL mutations may appear in HSCs, supporting the idea that CLL pathogenic events occur at an early stage of the hematopoietic process [18]. Taking the previous studies in this field into account, it is well known that chromosomal abnormalities as well as gene mutations are important events in CLL pathogenesis [19].However, it is still not clear which genetic events are related with the origin of the disease and when these alterations occur and have a functional impact inducing tumoral cell expansion during B cell differentiation.For these reasons, in this study, chromosomal abnormalities and gene mutations in hematopoietic progenitors were analyzed, showing that the whole population of CD34+ progenitors, even at the level of CD34 +CD19-, are already affected at genetic level in CLL patients.In particular, mutations of FBXW7, MYD88, NOTCH1, and XPO1 as well as 11q and 13q deletions were detected in CD34+ progenitors.By contrast, the origin of TP53 and SF3B1 mutations and IGH alterations could take place at a later maturational stage.Apart from B lymphocytes, some of these genetic alterations were also observed in other mature cell fractions (T lymphocytes and monocytes) derived from HSCs.Integrating all these results, a pattern of appearance and expansion of these genetic events during B-CLL cell differentiation was suggested. ",
"section_name": "Background",
"section_num": null
},
{
"section_content": "",
"section_name": "Methods",
"section_num": null
},
{
"section_content": "Samples were collected from the bone marrow (BM) of 56 CLL patients.CLL was diagnosed according to the World Health Organization (WHO) classification [20] and the National Cancer Institute (NCI) Working Group criteria [21].A complete immunophenotypic analysis of all cases was carried out by flow cytometry.The main biological features of the CLL patients are summarized in Additional file 1: Table S1. ",
"section_name": "Patients",
"section_num": null
},
{
"section_content": "Total CD34+ progenitor cells and CD19+ B cells were separately isolated from BM samples of CLL patients using magnetically activated cell sorting (MACS) CD34 and CD19 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany), respectively, according to the manufacturer's instructions.The workflow followed consisted of three steps: first, the isolation of the whole population of CD34+ cells (including CD34+CD19-early progenitors and CD34+CD19+ pro-B cells) from the total BM mononuclear cells, followed by the selection of CD19+ cells from the CD34 negative cell fraction resultant from the first step.Cell purities were determined by flow cytometry, being greater than 90 and 98% for each CD34+ and CD19+ cell fractions, respectively. In addition, fluorescence-activated cell sorting (FACS) (BD Biosciences, San Jose, CA, USA) was carried out in order to sort the specific subpopulation of CD34+CD19 cells as well as other mature cells such as CD19+ B lymphocytes, CD3+ T lymphocytes, and CD14+ monocytes, from peripheral blood (PB) samples in a second time point of the disease of seven CLL patients.Samples were stained with FITC anti-CD14 (Beckman Coulter), phycoerythrin (PE) anti-CD3 (Becton Dickinson), PE-Cy7 anti-CD19 (Immunostep S.L.), PerCP-Cy5.5 anti-CD45 (BioLegend), and allophycocyanin (APC) anti-CD34 (Becton Dickinson).Purities were greater than 98% in all cell fractions (Additional file 1: Figure S1). Genomic DNA was extracted from the different cell populations by column-based purification (AllPrep DNA/ RNA Mini Kit, Qiagen, Hilden, Germany) following the manufacturer's instructions. ",
"section_name": "Cell isolation and DNA extraction",
"section_num": null
},
{
"section_content": "NGS was performed in CD19+ B lymphocytes from all 56 CLL patients.Amplicon-based NGS was carried out on a GS Junior platform (454 Life Sciences, Branford, CT, USA) using the 454 Titanium Amplicon system (Roche Applied Science, Penzberg, Germany) [22] to investigate the mutational status of TP53 (exons 4-11), NOTCH1 (exons 33-34), SF3B1 (exons 10-16), FBXW7 (exons 8-12), MYD88 (exons 4-5), and XPO1 (exons 14-15) in CD19+ cells.The mutations identified in CD19+ cells were further analyzed in the corresponding whole population of CD34+ progenitors in order to determine whether the same mutations were present in an earlier step than B mature cells.Primer information, PCR conditions, and oligonucleotide design used in previous studies were adopted [23,24].The oligonucleotide was designed as part of the work of the IRON-II network.Sequencing data were obtained and analyzed using the GS Data Analysis Software package (Roche Applied Science, Penzberg, Germany) and the Sequence Pilot software for genetic analysis (JSI Medical Systems, Ettenheim, Germany).Mutations detected in more than 2% of bidirectional reads per amplicon in CD19+ cells and in more than 10% in CD34+ cells were accepted taking into account sequencing coverage (median 980 reads; coverage range 304-9387-fold) [25,26] and MACS purities from each cell population (98% for CD19+ and 90% for CD34+ cells). ",
"section_name": "Next-generation sequencing",
"section_num": null
},
{
"section_content": "To define if the mutations appeared in the specific subpopulation of CD34+CD19-cells and other mature populations derived from the hematopoietic progenitors, mutated target regions were sequenced by ultra-deep NGS, using an Illumina platform, in flow-sorted CD34 +CD19-, CD19+, CD3+, and CD14+ cell populations from seven CLL patients.NGS analysis was performed on MiSeq (Illumina, San Diego, CA, USA) using genomic DNA from peripheral blood flow-sorted CD19+ B lymphocytes, CD34+CD19-early progenitors, CD3+ T lymphocytes, and CD14+ monocytes.DNA was amplified using REPLI-g Mini Kit (Qiagen, Hilden, Germany).Target PCRs were performed using exon-specific primers (Additional file 1: Table S2).The experimental design and reaction conditions followed the manufacturer's recommendations.Briefly, PCR products were purified with High Pure PCR Product Purification Kit (Roche Diagnostics, Mannheim, Germany) and quantified using Qubit dsDNA HS Assay Kit (Life Technologies, Waltham, MA, USA).The purified amplicons were pooled to a total amount of 50 ng.The indexed paired-end library was prepared with NEBNext Ultra II DNA Library Prep kit for Illumina (NEW ENGLAND BioLabs) and sequenced using MiSeq (median coverage 4399 reads; range 1491-8614-fold).In order to verify the accuracy of the variant allele frequency (VAF), non-amplified DNA was sequenced in all cases with available material, finding no differences comparing to the VAFs obtained with a previous step of whole-genome amplification. In-house pipeline was performed to analyze sequencing data.Sequencing reads were aligned to the reference genome GRCh37/hg19 using BWA-0.7 [27].The alignments were refined with tools of the GATK-3.5 suite [28], and the variants were called according to GATK Best Practice recommendations [29,30].Finally, ANNOVAR was used for annotations and prediction of functional consequences [31]. The variant detection was set at 2% taking into account the sequencing coverage and the purities from all sorted cell fractions (more than 98%).Mutations detected at low frequencies (< 15%) by ultra-deep NGS were also validated using 454 Titanium Amplicon System (Roche Applied Science, Penzberg, Germany) (median 1712 reads; coverage range 1277-2638-fold) [23,24]. ",
"section_name": "Ultra-deep NGS",
"section_num": null
},
{
"section_content": "Interphase FISH was carried out in B cells from 56 BM samples using commercially available probes: 11q22/ ATM, 12p11.1-q11(alpha satellite), 13q14, 14q32/IGH, and 17p13/TP53 (Vysis/Abbott Co, Abbott Park, IL, USA).Dual-color FISH using differently labeled control and test probes was implemented following the methods previously described [32].FISH was also performed in the CD34+ cells of a group of nine CLL patients to assess the presence of the cytogenetic alterations identified in the corresponding CD19+ cell fraction.Samples were placed in a Cytospin cytocentrifuge (Thermo Scientific, Waltham, MA, USA) to concentrate the low number of cells.Signal screening was performed in at least 200 cells with well-delineated fluorescence spots.According to our cut-off standards, a score ≥ 10% was considered positive in all cases. ",
"section_name": "Fluorescence in situ hybridization",
"section_num": null
},
{
"section_content": "Statistical analyses were performed using IBM SPSS for Windows, Version 22.0 (IBM Corp., Armonk, NY, USA).Time to first therapy (TFT) and overall survival (OS) were analyzed on the date of the initial FISH study.Only leukemia-related deaths were considered when analyzing OS.The chi-square test was used to assess associations between categorical variables; continuous variables were analyzed with the Mann-Whitney U test.Variables significantly associated with TFT and OS were identified by the Kaplan-Meier method, and the curves of each group were compared with the log-rank test.Results were considered statistically significant for values of p < 0.05. ",
"section_name": "Statistical analysis",
"section_num": null
},
{
"section_content": "",
"section_name": "Results",
"section_num": null
},
{
"section_content": "Sequencing studies revealed a total of 28 mutations in 24 of the 56 (42.9%)CLL patients.Most of these patients (20/24; 83.3%) showed a single mutation in the analyzed genes, and four of them had two mutations in different genes (ID-34, ID-49, ID-50, and ID-53) (Table 1).The most frequently mutated gene was NOTCH1 (23.2%), followed by XPO1 (8.9%), SF3B1 (7.1%), FBXW7 (5.4%), TP53 (3.6%), and MYD88 (1.8%) (Fig. 1).All of them have been previously reported as mutations in the COS-MIC database (http://cancer.sanger.ac.uk/cosmic).All patients with mutations in NOTCH1 carried the same alteration (p.P2514Rfs*4), while XPO1 mutations corresponded to a previously reported gain-of-function mutation (p.E571K) in all cases.In addition, all SF3B1, FBXW7, TP53, and MYD88 mutations analyzed were missense mutations. In order to assess whether the mutations identified in CD19+ cells were also present in a previous step during B cell differentiation, the mutated target regions were analyzed by NGS in the total CD34+ cells.Strikingly, 15/ 24 patients (62.5%) showed the same mutations in their corresponding CD34+ cells (Table 1).The allele frequencies of mutations observed in a higher percentage than 10% of both CD19+ and CD34+ cell populations were compared calculating a CD19/CD34 ratio based on the percentage of mutated cells from each cell population.The cut-off CD19/CD34 ratio of 2.5 revealed two different mutational patterns between both cell fractions: \"maintained\" (ratio < 2.5) and \"decreased\" (ratio ≥ 2.5).Specifically, most of the mutations in NOTCH1 (7/9) and XPO1 (4/4) presented a similar mutational burden in both CD19+ and CD34+ cell fractions (Table 1; Fig. 2a).By contrast, alterations in TP53 (2/2), FBXW7 (2/2), and SF3B1 (3/4) showed a clearly lower percentage or even an absence in the CD34+ cells with respect to the corresponding mature B lymphocytes (Table 1; Fig. 2b). In a further step to assess if the mutations observed in the whole population of CD34+ progenitor cells appeared in the subpopulation of CD34+CD19-early hematopoietic progenitors, ultra-deep NGS was performed.Flow-sorted CD34+CD19-cells in a second time point from PB of a subset of patients were sequenced, confirming that six out of seven mutations-validated on the B lymphocytes from this time point-were also detected in the hematopoietic progenitor cells (Table 2).Particularly, MYD88, NOTCH1, XPO1, and FBXW7 mutations were observed in CD34 +CD19-cells.On the other hand, SF3B1 mutation was not observed in the CD34+CD19cells from patient ID-50.Apart from this, this patient, who was treated before the second time point, did not show FBXW7 mutation in its B lymphocytes. ",
"section_name": "Mutations of driver genes are already present in hematopoietic progenitor cells of CLL patients",
"section_num": null
},
{
"section_content": "Ultra-deep NGS revealed that gene mutations can be also present in other mature cells derived from hematopoietic stem cells.Thus, the same mutations detected in CD19+ B lymphocytes as well as in their corresponding CD34+CD19 progenitors were also detected in a very low percentage of CD3+ cells and in CD14+ cells in some CLL patients (Table 2).Specifically, ID-13 and ID-36 patients, who harbored MYD88 and FBXW7 mutations, respectively, also presented these mutations in both CD3+ and CD14+ cell populations.Moreover, ID-57 (FBXW7 mutated) showed the mutation on its monocytes whereas ID-37 (XPO1 mutated) carried the same alteration on the T lymphocytes. Interestingly, when the allele frequencies from all cell populations were compared, different patterns could be observed.First, MYD88 and NOTCH1 mutations (ID-13 and ID-42) appeared in > 10% of CD34+CD19-cells.In case of XPO1 and FBXW7, their mutations also appeared in CD34+CD19-cells but in a relatively low percentage (< 5%).In addition, NOTCH1 mutations only appeared on the hematopoietic progenitors and the mature B lymphocytes whereas FBXW7 and MYD88 mutations Only cases with > 10% CD34+ cells mutated were considered providing the purity for this cell fraction was higher than 90% in all the cases seemed to appear in all the sequenced cell fractions, affecting even myeloid lineage and T lymphoid lineage. On the other hand, patient ID-50 did not present its SF3B1 mutation in the CD34+CD19-nor the T lymphocytes and monocytes (Table 2).All these mutations detected at low frequencies (< 15%) in CD34+CD19-, CD3+, and CD14+ cells were also validated by 454 sequencing when material was available (Additional file 1: Table S3). Several cytogenetic abnormalities are detected in a previous developmental stage of the mature B lymphocytes of CLL patients FISH studies revealed a total of 39/56 (69.6%)CLL patients with cytogenetic abnormalities in B lymphocytes.Specifically, 13q deletion was the most common aberration in our cohort (46.3%), followed by trisomy 12 (17%), 11q deletion (11.3%),IGH alterations (9.3%), and 17p deletion (1.9%) (Additional file 1: Table S1). Fig. 1 Cytogenetics and molecular characteristics of CD19+ and CD34+ cells.In the heatmap, rows correspond to the indicated alterations and each column represents individual CLL samples.Color-coded based on the gene and cytogenetic status (dark gray, altered; light gray, not-altered; white, not analyzed).For IGHV status: dark gray, unmutated; light gray, mutated.*Only mutations with VAF > 10% were considered for MACS isolated CD34+ cells considering the purities obtained.**Mutations in CD34+CD19-cells were assessed by ultra-deep NGS, considering mutations with VAF > 2%, taking into account the cell purities obtained from FACS sorting To asses if the chromosomal abnormalities where also present in a previous step of the B cell differentiation, FISH analyses were performed in the whole population of CD34+ cells of a subset of patients (n = 9) (Fig. 1).Interestingly, these analyses revealed that five of nine CLL patients with cytogenetic alterations in mature B lymphocytes showed the same chromosomal aberration in the CD34+ cells, although at a lower percentage than in CD19+ cells (Table 3).Specifically, both CLL patients with 11qand three of five patients with 13q-showed the same cytogenetic alteration in the corresponding CD34+ cells.By contrast, IGH abnormalities identified by FISH in two CLL patients (ID-02 and ID-17) were not identified in their corresponding CD34+ cells.Apart from these alterations, the only CLL patient with +12, whose CD34+ cells were analyzed by FISH, did not have this trisomy in their corresponding progenitor cells. ",
"section_name": "Distinctive pattern of distribution of CLL driver mutations along hematopoietic lineages",
"section_num": null
},
{
"section_content": "The combination of NGS and FISH data revealed that two patients presented both mutations and chromosomal abnormalities (Table 3).The first case (ID-02) had an IGH alteration and a NOTCH1 mutation in the CD19+ cells.However, FISH studies and NGS analysis of CD34+ progenitors revealed that the NOTCH1 mutation was the only genetic event.The second case (ID-21) showed two chromosomal abnormalities (11q-and 13q-) and an XPO1 mutation.These three alterations were also observed in the CD34+ cells, although 11q-and 13q-were present in a higher percentage of cells (72 and 49%) than the XPO1 variant (30%). Four cases showed a co-occurrence of mutations in two different genes.Thus, two patients (ID-34 and ID-49) carried mutations in NOTCH1 and XPO1, present in both CD19+ and CD34+ cell fractions, with NOTCH1 The cut-off set for the second time point was 2% (bold) provided that our FACS purities were higher than 98% in all cases for all cell populations ",
"section_name": "Patients with multiple genetic alterations show a hierarchy in the appearance of these events",
"section_num": null
},
{
"section_content": "The clinical impact of the presence of mutations in the whole population of CD34+ cells was explored.The presence of mutations in the whole CD34+ cell population was associated with an unmutated IGHV status (p = 0.003) and high levels of serum β 2 microglobulin (p = 0.011) (Additional file 1: Table S4).Interestingly, patients with mutations in their CD34+ progenitors also showed shorter OS (p = 0.01) (Additional file 1: Figure S2A) than patients without mutations in this cell fraction.In addition, patients harboring mutations in NOTCH1, SF3B1, and TP53 in their hematopoietic progenitors presented a shorter TFT (p = 0.028) (Additional file 1: Figure S2B).Moreover, comparing the two mutational patterns identified in the CD34+ cells fraction, the mutations maintained on the CD34+ progenitors were significantly associated with the absence of trisomy 12 in the B lymphocytes (p = 0.014) (Additional file 1: Table S5). Comparing the mutational burden in the CD34+ fraction between BM samples collected before and after treatment revealed no significant differences (p = 0.605).Four patients who received treatment before the extraction of the BM relapsed.Interestingly, all of them showed mutations in the CD34+ progenitors with a similar mutational burden as the corresponding CD19+ B lymphocytes (Additional file 1: Table S6). ",
"section_name": "Clinical and biological correlations with genetic alterations in CD34+ cells in CLL patients",
"section_num": null
},
{
"section_content": "Our data provide evidence of the presence of mutations and chromosomal abnormalities in early hematopoietic progenitors in BM samples of CLL patients.These results shed light on the cell of origin of CLL in a previous developmental stage to mature B cells, demonstrating CLL patients can also show genetic events in the CD34+ hematopoietic progenitors.These results are consistent with the findings of two recent studies [17,18].MACS isolation was performed on the whole population of CD34+ cells including CD34+CD19+ pro-B cells.A previous study has reported that the pro-B cell population is larger in the bone marrow of CLL patients than in healthy donors (mean range of 18% pro-B cells in the total bone marrow CD34+ cell count, exceeding 30% in some cases) [17].This suggests that the patients who presented a mutational burden of < 30% in the CD34+ cell population may only harbor the mutation in CD34 +CD19+ pro-B cells, rather than in the early hematopoietic progenitors.It is of particular note that 10 out of 15 patients with mutations in the whole population of CD34+ cells had mutational rates of > 30%, suggesting that these mutations not only appear in the pro-B cells, but also at earlier maturational stages of B cell differentiation (CD34+CD19-progenitors).In order to assess this hypothesis, we could perform ultra-deep NGS studies of flow-sorted CD34+CD19-cells using PB samples in a small subset of patients within the main cohort, detecting that all the mutations, except to one, were already present in this cellular fraction.Therefore, these results confirmed that mutations on CLL driver genes could occur in early hematopoietic progenitor cells of these patients.In particular, our sequencing results suggest that mutations in NOTCH1, MYD88, FBXW7, and XPO1 may appear in CD34+CD19-cells whereas TP53 and SF3B1 mutations could appear in a later stage of B cell differentiation.As far as we are concerned, these results were demonstrated for the first time in fresh hematopoietic progenitor cells without having been cultured. As these driver mutations have been detected in early hematopoietic progenitors of some CLL patients, we hypothesized that these alterations can affect hematopoietic lineages other than B cells.As it was previously reported, some mutations in well-known CLL drivers can also appear on a low percentage in other mature cell fractions as CD3+ T lymphocytes and/or CD14+ monocytes [18]. In order to determine the stage of B cell differentiation in which these mutations induced an expansion of the tumoral cell population, first, the mutational burdens in CD19+ mature B lymphocytes and CD34+ progenitors were compared.Since the mutational burden in CD34+ cells with NOTCH1 mutations was as high as that observed in CD19+ cells in most of the cases, it could be hypothesized that mutations in this gene induced an expansion of the CLL hematopoietic progenitors.Indeed, ultra-deep NGS studies confirmed this in patient ID-42 who had NOTCH1 mutation in more than 10% of CD34 +CD19-cells.Moreover, every XPO1 mutation observed in our cohort was present in the CD34+ progenitors and the mutational burden remained similar in both cellular fractions.However, the percentage of XPO1 mutations in the CD34+ cells exceeded 30% in very few cases, suggesting that these mutations could be enriched at an intermediate B cell stage as CD34+CD19+ pro-B cells.On the other hand, the mutational burdens of TP53, SF3B1, and FBXW7 were considerably lower in the CD34+ cell population.Although SF3B1 mutation was not detected in CD34+CD19-cells, suggesting it as a late event in B-CLL differentiation, one out of four SF3B1 mutated patients (ID-43) carried a mutation in a high percentage of the whole population of CD34+ progenitors, similar to results from a previous study [18].Besides this, alterations in this gene have been also reported in CD34+ cells of patients with myeloid malignancies [33].Therefore, it should be essential to study larger cohorts of CLL patients in order to determine what type of SF3B1 mutations occur in HSCs of CLL patients and functional studies to assess the differences between \"CLL-HSCs SF3B1 mutated\" and \"MDS-HSCs SF3B1 mutated.\" Focusing on the presence of cytogenetic abnormalities in CD34+ cell populations in our cohort, 11q-and 13qappeared in the CD34+ progenitors at high percentages, as reported previously [15,16], supporting the hypothesis that these chromosomal aberrations could be an early event in CLL [8].By contrast, IGH alterations were not present in any of the CD34+ hematopoietic progenitors.Previous case report studies have yielded similar results [34,35], suggesting that IGH alterations occur in an advanced stage of the lymphocyte maturation process. The analysis of patients with more than one genetic alteration allowed us to define a hierarchy of the appearance of these genetic events.When IGH alterations and the NOTCH1 mutation are present in the same patient (ID-02), it is clear that the NOTCH1 mutation is an earlier step than the IGH alteration during B cell differentiation.Moreover, when 11q-, 13q-, and XPO1 mutations are present in the same patient (ID-21), they all appear in the CD34+ progenitors.However, 11q-and 13q-are certainly present at a higher allele frequency than the mutational load of XPO1, suggesting that cells with 11qand 13q-were expanded in an earlier stage than the XPO1 mutation in the pathogenesis of the disease [8].Specifically, the dominant clone in the two cases with a double mutation in NOTCH1 and XPO1 (ID-34 and ID-49) was always NOTCH1 rather than XPO1.Therefore, since XPO1 is still present in a small part of the CD34 +CD19-cell population and greatly enriched on the total CD34+ fraction, we may consider that cells carrying XPO1 mutations are expanded in an intermediate event of B-CLL differentiation, given the previous possible events such as 13q-and 11q-abnormalities or MYD88 and NOTCH1 mutations.Taking into account all these results, a model of the appearance of genetic events during the hematopoiesis in CLL has been suggested (Fig. 3).However, as some of these genetic alterations were observed in few cases, it would be interesting to sequence all cell fractions in larger cohorts of patients. The prognostic impact of gene mutations and chromosomal abnormalities in CLL has been well characterized in several studies of large cohorts of patients [4,9,23,36].TP53, NOTCH1, and SF3B1 are described as poor prognostic mutations [10,[37][38][39][40].The prognostic impact of the presence of mutations in CD34+ cells from CLL patients has been assessed for the first time in this study, showing that CLL patients harboring mutations in the hematopoietic progenitors showed worse prognosis.It is essential to achieve a better understanding of these results since only six genes were analyzed in our study, whereas a CLL exome exhibits an average of 20 mutations [8,10].Therefore, further studies analyzing the whole exome in larger cohorts should be performed in order to accurately define their impact.In addition, conventional CLL therapies may not be able to eradicate or reduce the malignant CD34 + CLL clone and may be responsible for the relapse of patients bearing mutations in these cells.Given that these therapies seem not to have a clear effect on CD34+ CLL cells, allogeneic stem cell transplantation could be an option for overcoming the challenges that may arise from CD34+ cell treatment [41]. ",
"section_name": "Discussion",
"section_num": null
},
{
"section_content": "Our data show that recurrent CLL chromosomal abnormalities and gene mutations are present not only in mature B lymphocytes but also in hematopoietic progenitors.Although CLL is a clonal mature B cell disease, our results provide strong evidence that CLL may originate in the early stages of hematopoiesis.Both chromosomal alterations and point mutations are highly relevant to the disease pathogenesis, with a clinical impact as soon as they appear.To the best of our knowledge, our study is the first to analyze both genetic events in different cellular fractions of the same patients by NGS and FISH.It provides us with a broader understanding of CLL initiation and development, opening up possibilities for future therapies. ",
"section_name": "Conclusions",
"section_num": null
},
{
"section_content": "Additional file 1: Table S1.Clinico-biological characteristics from CLL patients.Table S2.Illumina Primer Design.Table S3.Validation of mutations detected at low frequency by ultra-deep NGS in flow-sorted cell fractions using 454 sequencing.Table S4.Patients' characteristics regarding the presence of mutations in CD34+ progenitors.Table S5.Patients' characteristics regarding the mutational burden maintenance or decrease in CD34+ progenitors.Table S6.Treatments prior bone marrow extraction of 4 CLL patients who relapsed and correlation with the CD19 + and CD34+ mutational status. ",
"section_name": "Additional file",
"section_num": null
}
] |
[
{
"section_content": "We thank Irene Rodríguez, Sara González, Teresa Prieto, M a Ángeles Ramos, Almudena Martín, Ana Díaz, Ana Simón, María del Pozo, Isabel M Isidro, Vanesa Gutiérrez, Sandra Pujante, and Sandra Santos from the Centro de Investigación del Cáncer, Salamanca, Spain, for their technical assistance. ",
"section_name": "Acknowledgements",
"section_num": null
},
{
"section_content": "This work was partially supported by grants from the Spanish Fondo de Investigaciones Sanitarias PI12/00281, PI15/01471, Instituto de Salud Carlos III (ISCIII), European Regional Development Fund (ERDF) \"Una manera de hacer Europa,\" Consejería de Educación, Junta de Castilla y León (SA085U16), Proyectos de Investigación del SACYL, Spain: GRS 1172/A/15, BIO/SA10/14, GRS 1343/A/16, and Fundación Española de Hematología y Hemoterapia (FEHH) and by a grant (RD12/0036/0069) from the Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness & European Regional Development Fund (ERDF) \"Una manera de hacer Europa\" (Innocampus; CEI-2010-1-0010), Fundación \"Memoria Don Samuel Solórzano Barruso\" 2016, and the European Union Seventh Framework Programme [FP7/2007-2013] under Grant Agreement no.306242-NGS-PTL.MHS is fully supported by an \"Ayuda predoctoral de la Junta de Castilla y León\" by the Fondo Social Europeo (JCYL-EDU/346/2013 PhD scholarship).Oligonucleotide primer plates for amplicon deep-sequencing were provided by Roche Diagnostics, Penzberg, Germany, as part of the IRON-II study. ",
"section_name": "Funding",
"section_num": null
},
{
"section_content": "The datasets analyzed 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": "Abbreviations BM: Bone marrow; CLL: Chronic lymphocytic leukemia; FACS: Fluorescenceactivated cell sorting; FISH: Fluorescence in situ hybridization; HSC: Hematopoietic stem cell; IGHV: Immunoglobulin heavy chain gene; MACS: Magnetically activated cell sorting; NGS: Next-generation sequencing; OS: Overall survival; PB: Peripheral blood; TFT: Time to first therapy; VAF: Variant allele frequency Authors' contributions MQÁ and MHS designed and performed the research and statistical analyses, analyzed the data, and wrote the paper.CR and RB performed the nextgeneration sequencing studies and analyzed the data.AERV designed the study and performed the cell isolation.JMHS and DQ performed the cell isolation and FISH studies.AM proceeded the samples and performed the sequencing studies.AAM, MJVM, GFG, MPDB, JG, JNR, GMN, JMA, AGC, JAQ, MS, and CA provided the patients' data.JAH provided the patients' data and critically reviewed the manuscript.MGA performed the IGHV mutational status analysis.AK designed the sequencing studies.MG designed the IGHV mutational status analysis and critically reviewed the manuscript.JAH critically reviewed the manuscript.JMHR designed and performed the research and corrected and approved the final version of the manuscript.All the authors approved the final version of manuscript. The authors declare that they have no competing interests. Not applicable. The present study was approved by the local ethics committee (Comité Ético de Investigación Clínica, Hospital Universitario de Salamanca).Written informed consent was obtained from all participants before they entered the study. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Abbreviations BM: Bone marrow; CLL: Chronic lymphocytic leukemia; FACS: Fluorescenceactivated cell sorting; FISH: Fluorescence in situ hybridization; HSC: Hematopoietic stem cell; IGHV: Immunoglobulin heavy chain gene; MACS: Magnetically activated cell sorting; NGS: Next-generation sequencing; OS: Overall survival; PB: Peripheral blood; TFT: Time to first therapy; VAF: Variant allele frequency Authors' contributions MQÁ and MHS designed and performed the research and statistical analyses, analyzed the data, and wrote the paper.CR and RB performed the nextgeneration sequencing studies and analyzed the data.AERV designed the study and performed the cell isolation.JMHS and DQ performed the cell isolation and FISH studies.AM proceeded the samples and performed the sequencing studies.AAM, MJVM, GFG, MPDB, JG, JNR, GMN, JMA, AGC, JAQ, MS, and CA provided the patients' data.JAH provided the patients' data and critically reviewed the manuscript.MGA performed the IGHV mutational status analysis.AK designed the sequencing studies.MG designed the IGHV mutational status analysis and critically reviewed the manuscript.JAH critically reviewed the manuscript.JMHR designed and performed the research and corrected and approved the final version of the manuscript.All the authors approved the final version of manuscript. ",
"section_name": "",
"section_num": ""
},
{
"section_content": "The authors declare that they have no competing interests. ",
"section_name": "Competing interests",
"section_num": null
},
{
"section_content": "Not applicable. ",
"section_name": "Consent for publication",
"section_num": null
},
{
"section_content": "The present study was approved by the local ethics committee (Comité Ético de Investigación Clínica, Hospital Universitario de Salamanca).Written informed consent was obtained from all participants before they entered the study. ",
"section_name": "Ethics approval and consent to participate",
"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
}
] |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.